July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
A Small Molecule Screen in Zebrafish to Identify Mediators of Retinal Neovascularization
Author Affiliations & Notes
  • Donald Wayne Van Fossan
    Ophthalmology, UCSD, La Jolla, California, United States
  • Footnotes
    Commercial Relationships   Donald Van Fossan, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4891. doi:
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      Donald Wayne Van Fossan; A Small Molecule Screen in Zebrafish to Identify Mediators of Retinal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4891.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Age-related Macular degeneration (AMD) and diabetic retinopathy are leading causes of blindness worldwide. At the heart of both of these diseases are retinal vascular leakage and neovascularization of retinal and choroidal blood vessels. While the development of intravitreal injection of inhibitors of vascular endothelial growth factor has revolutionized treatment for these diseaes, there are still over 40% of patients who do not have complete treatment response. Thus, an unmet need is to identify other mediators of retinal neovascularization which are independent of the VEGF pathway which may help to improve treatment for AMD and diabetic retinopathy. Previously it has been shown that zebrafish vhl mutants show aberrant retinal and choroidal neovascularization and vessel leakage. We are exploiting the small size and scalability of the zebrafish system to perform a high throughput small molecule screen in an intact in vivo animal to look for small molecules which can inhibit retinal neovascularization in zebrafish vhl mutants.

Methods : Single cell zebrafish embryos with the fli-1:GFP background were injected with Cas9 protein and 2 vhl gRNAs to generate vhl -/- mutants. Vhl crispr injected fish robustly display the vhl mutant phenotype with high penetrance. Fish were screened for the vhl -/- phenotype at 3 days post-fertilization (dpf) and loaded into the wells of 96-well plates with small molecules from the Tocris plus library (Tocris, Minneapolis MN) at 10 uM concentration. At 5.5 dpf, the intersegmental vessels were then scored using a fluorescent microscope to determine if the vhl -/- phenotype was still present or if it was rescued. 4 uM Sunitnib was used as a positive control to verify rescue of neovascularization. Rescued phenotypes were then screened in a secondary trial for similar results. Any fish the showed positive results in the secondary screen were then further studied for their potential role in rescuing the vhl -/- phenotype and providing potential anti-angiogenic affects.

Results : 1280 molecules from the Tocris plus library were screened. (x) molecules demonstrated prevention of neovascularization of the ISV vessels. Confirmation and further characterization of these molecules will be presented.

Conclusions : The zebrafish vhl mutant can be a useful tool to identify small molecules to inhibit retinal neovascularization

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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