Targeting Ocular Neovascularization with Novel APE1/Ref‐1 Inhibitors

Abstract

Background and ObjectivePathological ocular neovascularization is implicated in several blinding eye diseases such as retinopathy of prematurity, diabetic retinopathy, and wet or exudative age related macular degeneration (AMD). Wet AMD is characterized by choroidal neovascularization, where friable vessels grow under the retina causing distortion of the normally flat macula and loss of central vision. Apurinic/apyrimidinic endonuclease 1/Redox effector factor‐1 (APE1/Ref‐1 or Ref‐1) serves dual functions as a DNA repair protein and a redox‐sensitive transcriptional regulator. Key proangiogenic transcription factors such as HIF1α, STAT3 and NFκB are under the regulation of Ref‐1, hence its inhibition is a potential tool in the fight against pathological neovascularization. We investigated whether novel, second generation small molecule Ref‐1 inhibitors, APX2009 and APX2014, can ameliorate pathological ocular neovascularization.MethodsWe tested the inhibitors for antiproliferative effect on Human Retinal Microvascular Endothelial primary cells (HRECs) and the Rhesus choroidal endothelial cell line (Rf/6a). We then assessed their ability to affect endothelial cell properties of tubule formation in vitro. Cells treated with compound or DMSO were seeded on solidified Matrigel to assay their ability to form vascular tubes, and the scratch‐wound assay was also used to test migration, another important endothelial property. We then examined both compounds ex vivo in a choroidal sprouting assay, which models microvascular angiogenesis from tissue. Finally, we tested APX2009 in vivo in the laser‐induced choroidal neovascularization (L‐CNV) mouse model, which recapitulates features of wet AMD. Mice were treated with APX2009 twice daily via intraperitoneal injection after laser until 14 days post treatment, when neovascularization was quantified by isolectin staining.ResultsAPX2014 was more antiproliferative in both HRECs (GI50: 110 nM) and Rf/6a (GI50: 5 μM) than APX2009 (HREC GI50: 1.13 μM; Rf/6a GI50: 25.7 μM). Both compounds significantly reduced the ability of both HRECs and Rf/6a cells to form tubes at mid nanomolar concentrations compared to DMSO. Both APX2009 and APX2014 significantly inhibited HREC and Rf/6a cell migration into the scratched area at the lowest concentrations tested. Ex vivo, both APX2009 and APX2014 inhibited angiogenic sprouting at low micromolar and high nanomolar concentrations respectively. In vivo, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4‐fold compared to vehicle (p<0.0001, ANOVA with Dunnett's post hoc tests), effectively treating L‐CNV, without obvious intraocular or systemic toxicity.ConclusionRef‐1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo. In addition, APX2009 is an effective systemic therapy for CNV in vivo, thus establishing Ref‐1 inhibition as a promising therapeutic route to treat ocular neovascularization.Support or Funding InformationNIH/NEI R01EY025641, Research to Prevent Blindness, Apexian PharmaceuticalsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.