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Breandan N Kennedy, Stephanie Merrigan, Eugene Dillon, Gerard Cagney, Sally Hampton; Phenotypic Identification of Vitamin D Receptor Agonists as Regulators of Developmental Angiogenesis and miR21 in the Zebrafish Eye. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3598.
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© ARVO (1962-2015); The Authors (2016-present)
Angiogenesis is fundamental for development and problematic in disease. Ocular disorders including neovascular age related macular degeneration and proliferative diabetic retinopathy have an aberrant angiogenesis component. Our in vivo unbiased screen of 465 ICCB library chemicals identified inhibitors of developmental angiogenesis in the zebrafish eye. Such small molecule inhibitors give insight into regulators of angiogenesis and represent potential anti-angiogenic treatments. Here, we followed up on the safety and efficacy of lead hit calcitriol.
Calcitriol and vitamin D receptor agonists (VDRA) were tested for inhibition of ocular hyaloid vessel (HV) and non-ocular inter-segmental vessel (ISV) developmental angiogenesis in zebrafish larvae. Safety studies in larvae used light microscopy to evaluate retinal morphology and the optokinetic response (OKR) assay to assess visual function in response to calcitriol treatment. Mechanistic understanding was gained through proteomics, ELISA and QRT-PCR studies in treated zebrafish larval eyes and human retinal pigment epithelial cells (ARPE-19). The anti-angiogenic activity of calcitriol was further evaluated in an ex vivo C57BL/6J mouse model of choroidal sprouting angiogenesis.
Calcitriol and 7 VDRA significantly inhibited HV developmental angiogenesis by up to 50 percent (P〈0.001, N≥20). Absence of phenotypically observable change in pre-established and developing ISV indicated ocular selective anti-angiogenic activity (N≥15). Calcitriol treated larvae presented with diminished OKR despite appearance of normal retinal lamination/morphology in ocular cross sections (N≥27, P〈0.001)(N≥3). Treatment with 0.1-10 µM calcitriol induced miR21 expression and treatment with ≥10 µM calcitriol induced vegfaa expression in larval eyes (n=3). Proteomics of calcitriol treated larval eyes revealed 32 differentially regulated proteins (P〈0.05). Preliminary data suggests calcitriol to attenuate TNFα induced IL-8 expression in ARPE-19 cells (n=2). Anti-angiogenic efficacy was validated in the ex vivo mouse choroidal sprouting angiogenesis model (n=3).
VDRA regulate ocular developmental angiogenesis and miR21 expression in the zebrafish eye. VDRA and/or its downstream mediators such as miR21 signify potential targets for the treatment of neovascular diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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