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Stephanie Merrigan, Orla Galvin, Alison Reynolds, Breandan N Kennedy; Vitamin D Receptor Agonists Significantly Attenuate Ocular Developmental Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):158.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular angiogenesis underpins the vasculature leakage, scar formation and blindness in neovascular age related macular degeneration (nAMD). A clinical need exists to improve current nAMD therapies associated with repeated invasive injections, high costs and adverse drug reactions. To identify novel small molecule inhibitors of ocular angiogenesis, a phenotype-based screen of the Institute of Chemistry and Cell Biology (ICCB) Known Bioactives Library was performed in zebrafish and identified Calcitriol as a hit.
Compounds were screened for inhibition of hyaloid vessel (HV) angiogenesis in Tg(fli1:EGFP) zebrafish larvae from 2-5 days post fertilisation (dpf) and inhibition of inter-segmental vessel (ISV) angiogenesis between 6 hours post fertilisation and 2 dpf. The efficacy and potency of Vitamin D receptor agonists was determined in HV and ISV dose response assays. Larvae were fixed in 4% PFA and analysed by fluorescent microscopy. Safety pharmacology was assessed by light microscopy examination of the morphological integrity of larval retinal sections and cytotoxicity profiling of human retinal pigment epithelium cells (ARPE-19). An aortic ring angiogenesis assay with C57BL/6 mice was utilised to determine anti-angiogenic efficacy in a mammalian model.
Vitamin D receptor (VDR) agonists significantly inhibit vessel formation in the eye (HV) but not in the trunk/tail (ISV) (P< 0.0001) of zebrafish larvae. Tacalcitol followed by Calcifediol, Paricalcitol, Doxercalciferol, Calcipotriol, Calcitriol, EB 1089 and 22-Oxacalcitriol demonstrated 50-20% inhibition of HV angiogenesis. Calcitriol- or EB1089-treated (10 uM) larval eyes show slight oedema, normal lamination and no increased apoptosis. ARPE-19 cells show dose-dependent cytotoxicity to Calcitriol and EB1089 (P< 0.0001) with highest tolerated doses of 10 µM. Preliminary data shows Calcitriol, EB1089 and 22-oxacalcitriol to reduce aortic ring angiogenic sprouting.
Here, we demonstrate that VDR agonists significantly and specifically inhibit ocular angiogenesis during zebrafish development. Future experiments will evaluate the molecular mechanisms of VDR anti-angiogenic activity and the efficacy of VDR agonists in pre-clinical ocular neovascularisation models.
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