Ref-1/APE1 Inhibition with Novel Small Molecules Blocks Ocular Neovascularization

Abstract

Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-B and other proangiogenic transcription factors. An existing inhibitor of Ref-1's function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI APX2009: 1.1 M, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI APX2009: 26 M, APX2014: 5.0M). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-B activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle ( < 0.0001, ANOVA with Dunnett's post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.

Ferrochelatase regulates retinal neovascularization

“…In vivo OCT was performed in griseofulvin injected adult mice as described previously 39,42 at 5 and 14 days after injection using the Micron III intraocular imaging system (Phoenix Research Laboratories, Pleasanton, CA, USA). Briefly, before the procedure, mice were anesthetized with ketamine (90 mg/kg) and xylazine (5 mg/kg) and their pupils were dilated with 1% of tropicamide solution (Alcon, Fort Worth, TX, USA) and lubricated with hypromellose ophthalmic demulcent solution (Gonak; Akorn, Lake Forest, IL, USA).…”

Chrysin Inhibits High Glucose-Induced Migration on Chorioretinal Endothelial Cells via VEGF and VEGFR Down-Regulation

“…In this study, chorioretinal EC (RF/6A) was used as the main cell model for retinopathy and cultured under high glucose conditions to mimic the environment of high blood glucose. Although the endothelial characteristics of RF/6A cells were questioned [ 19 ], abundant literature shows that RF/6A cells are still used as endothelial cell lines to model retinal and choroidal angiogenesis [ 20 , 21 , 22 , 23 , 24 ]. Culturing ECs under high-glucose conditions cannot mimic all characteristics of DM but might explain the endothelial growth factor autocrine mechanism of the ECs [ 25 , 26 ].…”

Ferrochelatase regulates retinal neovascularization

“…In vivo OCT was performed in griseofulvin injected adult mice as described previously (39,42) at the indicated times using the Micron III intraocular imaging system (Phoenix Research Laboratories, Pleasanton, 7 CA, USA). Briefly, before the procedure, mice were anesthetized with ketamine (90 mg/kg) and xylazine (5 mg/kg) and their pupils were dilated with 1% tropicamide solution (Alcon, Fort Worth, TX, USA) and lubricated with hypromellose ophthalmic demulcent solution (Gonak; Akorn, Lake Forest, IL, USA).…”