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N. Sheibani, B. Gao, E. Mustafarj, E. A. Scheef; A Cell-Based High Throughput Screening Assay for Antiangiogenic Compounds. Invest. Ophthalmol. Vis. Sci. 2008;49(13):518. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Develop a cell-based high throughput screening assay for identification of compounds with antiangiogenic activity.
Using retinal endothelial cells (EC) isolated from thrombospondin-1-deficient (TSP1-/-) mice, we developed a scratch wound assay in the 96-well format. This assay takes advantage of the enhanced migratory phenotype of TSP1-/- retinal EC for identification of compounds with anti-migratory activity. Using this assay we screened a 16,000 compound library. The activities of the hit compounds were further confirmed by additional assays. We have identified a number of compounds with anti-migratory, anti-angiogenesis activity for in vivo screening.
Using our primary screening assay we identified approximately 400 compounds with significant anti-migratory activity in the scratch wound assay. We next limited our hits to those compounds that also resulted in reduced expression of fibronectin narrowing the number hits to 60 compounds. Using a transwell migration assay we identified 14 compounds with significant antimigratory activity. We have identified a few of these compounds that also show strong anti-angiogenic activity in the in vitro capillary morphogenesis and ex vivo aortic ring sprouting assays. We are presently confirming the anti-angiogenic activity of these compounds in the mouse model of oxygen-induced ischemic retinopathy.
We demonstrate the development and utilization of a cell-specific assay for high throughput screening and identification of compounds with antiangiogenic activity. Given the global role of TSP1 in regulation of angiogenesis, we believe the inhibitors identified here will have broad application against angiogenesis promoted by a variety of mediators.
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