Efficacy of echinomycin in hypoxia‐inducible factor‐mediated ocular angiogenesis

Abstract

AbstractPurposeEchinomycin (EKN), an inhibitor of hypoxia‐inducible factor (HIF)‐1 DNA‐binding activity, has been implied as a possible therapeutic agent in ischemic diseases. We assessed EKN in hypoxia‐driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE), and in vivo using the laser‐induced mouse choroidal neovascularization (CNV) model.MethodsAdult retinal endothelial were kept at normoxia (20% O2) or exposed to hypoxia (1% O2), in the presence or absence of EKN. Epithelial wound‐healing was used to determine the dose of EKN. Effects of EKN on hypoxia‐mediated pathways were analyzed by western blot for HIF‐1a, qPCR of HIF‐target genes, and proteome array for soluble angiogenic factors. Inhibition of angiogenesis by EKN was determined in human primary adult retinal endothelial cells (aREC), exposed to EKN‐treated hypoxic conditioned aRPE medium by spheroid sprouting assay. 8‐week‐old C57BL/6J mice underwent laser‐induced CNV, as a model of HIF‐associated ocular neovascularization. Animals were treated with EKN and compared to a mouse VEGFR1‐Fc chimera protein (equivalent of aflibercept). CNV lesion area was determined by fluorescein angiography.ResultsAdult retinal endothelial treated with EKN demonstrated hypoxia‐dependent decreased cell proliferation in the wound‐healing assay. Lower HIF‐mediated transcripts were detected in hypoxic aRPE cells treated with EKN compared with non‐treated controls. These results were confirmed by proteome profiler for angiogenic factors, and subsequently reduced hREC sprouting. Laser‐induced mice treated with 1 mg of intravitreally injected EKN demonstrated a significant decrease in vascular lesion area when compared with 1 mg of VEGFR1 chimera or vehicle‐treated controls.ConclusionsOur data suggest EKN as a potent inhibitor of HIF‐mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age‐related macular degeneration.