ZD6474 attenuates TGF-β1-induced fibrosis in human Tenon fibroblasts and inhibits neovascularization via AKT-mTOR signaling pathway

Purpose To investigate the anti-fibrotic effect of ZD6474 (a novel inhibitor of VEGF and EGF) in TGF-beta 1 stimulated human Tenon's capsule fibroblasts (HTFs) and the anti-angiogenetic role in HUVECs, compared to that of mitomycin C (MMC). Methods The effects of ZD6474 on cell proliferation or migration in TGF-beta 1-stimulated HTFs and HUVECs were determined, using CCK8 or wound healing assay, respectively. The typical markers of fibrosis in TGF-beta 1-stimuated HTFs were detected, vimentin by immunofluorescence, alpha-SMA and snail by western blot. Tube formation was applied to validate the anti-angiogenesis effect in HUVECs following ZD6474 treatment. Furthermore, phosphorylated AKT and mTOR (p-AKT and p-mTOR) were evaluated, compared to the standardized total AKT and mTOR, using western blot. Results There was almost no decreased cell viability in HTFs following ZD6474 (<= 1 mu M/mL) treatment, but MMC (> 50 mu g/mL) significantly impaired cell viability. ZD6474 significantly inhibited TGF-beta 1-stimulated proliferation and migration in HTFs, compared to control group (**P < 0.01). ZD6474 also significantly attenuated the TGF-beta 1-stimulated expression of vimentin, alpha-SMA and snail in HTFs. Tube formation was notably interrupted in HUVECs following ZD6474 treatment (**P < 0.01). P-AKT and p-mTOR were significantly decreased in response to ZD6474 treatment in TGF-beta 1- induced HTFs and HUVECs. Conclusions ZD6474 exerts anti-proliferation and anti-fibrotic effects in TGF-beta 1-stimulated HTFs perhaps via regulating AKT-mTOR signaling pathway. ZD6474 also inhibited proliferation, migration and tube formation in HUVECs via the same signaling pathway. We concluded that ZD6474 may be potentially a novel agent in preventing bleb dysfunction following glaucoma filtration surgery (GFS).