Abstract
Purpose: :
Glycogen synthase kinase-3β (GSK-3β), a downstream molecule of insulin signaling, plays an important role in glycogen metabolism, although only a few publications regarding angiogenic effects were reported. Here we investigated the effects of GSK-3β inhibitors on vascular endothelial growth factor (VEGF)-induced retinal angiogenesis.
Methods: :
Retinas from 7-8 week old C57BL/6J mice were isolated and cultured on Millicell filter with 25 ng/ml VEGF alone or VEGF + each dose of GSK-3β inhibitors. After 96 hours, retinas were fixed and applied to immunohistochemistry with PEACAM and collagen type IV, for the quantification of neovascular sprouts. Human umbilical vein endothelial cells (HUVECs) were cultured with CSC Complete Recombinant Medium (Cell Systems Corporation). Live cell imaging were performed using cytoplasmic staining of HUVECs with CellMaskTM, and confocal images were obtained at 0, 5, 15 min after the treatments. Phalloidin was used for the evaluation of actin cytoskeleton.
Results: :
GSK-3β inhibitors, SB216763 25 µM and AR-A014418 250 µM, decreased VEGF-induced neovascular sprouts in ex vivo retinal angiogenic model (p<0.05 and p<0.01, respectively). We additionally performed the timelapse imaging of HUVECs and found that cytoplasm at cell borders was retracted with the filopodia-like structure after the treatment with GSK-3β inhibitors, which might be compatible to their antiangiogenic effects. Immunostaining with phalloidin showed that actin stress fibers were produced 5 min after the treatment with GSK-3β inhibitors.
Conclusions: :
These data suggests GSK-3β inhibitors reduced retinal angiogenesis, mediated via cytoskeletal changes and concomitant cellular retraction, at least in part.
Keywords: neovascularization • retinal neovascularization