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O. S. Bond, N. S. Dejneka, J. M. Nigro, N. K. Shams; Identification of Human VEGF165 siRNAs that Spare VEGF165b. Invest. Ophthalmol. Vis. Sci. 2009;50(13):496.
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VEGF165b has been identified as an endogenous anti-angiogenic VEGF isoform. The goal of this study was to design an siRNA that selectively inhibits VEGF165 but spares VEGF165b.
ARPE19 cells were seeded in 24 well plates (50,000 cells per well). Eighteen to twenty-four hours post-seeding, cells were 50-75% confluent and used for transfection. Fourteen human VEGF165 specific siRNAs were designed and tested. Cells were transfected with the siRNAs (25 nM) using RibojuiceTM siRNA Transfection Reagent (Novagen) following the manufacturer’s protocol. Twenty-four hours post-transfection, media containing the transfection mixture was removed from the cells and replaced with serum free media containing 10 ng/mL human recombinant TGFβII. Twenty-four hours after TGFβII treatment, media was collected and analyzed for total VEGF protein via ELISA. A select number of siRNA candidates were put through an additional transfection screen. Cells were collected, RNA extracted, and semi-quantitative RT-PCR was performed to determine the siRNAs’ inhibitory effect on VEGF165, and VEGF165b. RT-PCR for GAPDH was used as a control.
Treatment of ARPE19 cells with TGFβII induced VEGF production in ARPE19 cells. ELISA results demonstrated several siRNA candidates inhibited the production of TGFβII-induced VEGF in ARPE19 cells. RT-PCR confirmed that 2 candidates inhibited production of VEGF165 but spared VEGF165b.
ARPE19 cells produce VEGF165b. siRNAs sparing VEGFA165b can be synthesized and may be more efficacious than siRNAs that knockdown both VEGF165 and VEGF165b isoforms. VEGF165b sparing siRNAs may be potent therapeutic candidates for the treatment of ocular neovascularization.
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