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Temitope Sasore, Alison Reynolds, Breandan Kennedy; EFFECTIVE TARGETING OF THE PI3K/AKT/mTOR PATHWAY: A PROMISING THERAPEUTIC OPTION FOR THE TREATMENT OF OCULAR NEOVASCULARIZATION/INFLAMMATION/OEDEMA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1939.
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There is a clinical need to develop improved pharmacological therapeutics for ocular neovascularization, retinal inflammation and retinal or oedema associated with diabetic retinopathy (DR), age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). In this study, we investigate the in vivo anti-angiogenic efficacy of a panel of PI3K/Akt/mTOR inhibitors, alone and in combination.
To measure the efficacy of inhibition of developmental angiogenesis, Tg(fli1:EGFP) transgenic zebrafish were treated with drugs from 1-5 post fertilisation and screened inhibitory effects on intersegmental vessel (ISV) and hyaloid vessel (HV) number. Visual motor response and optokinetic response were carried out to assess the effects on visual behaviour. The developmental expression pattern of PI3K subunits in the zebrafish examined using reverse transcriptase polymerase chain reaction. Effects on human endothelial cell number were examined using a crystal violet assay.
The broad-spectrum inhibitor LY294002, followed by the mTOR inhibitor rapamycin, ranked as the most effective PI3K pathway inhibitors of developmental angiogenesis in zebrafish. Additionally, there was significant anti-angiogenic effect exerted by the dual PI3K-mTOR inhibitors, NVP-BEZ235 and PI-103. Interestingly, some compounds that inhibit developmental angiogenesis of the HV, are ineffective at inhibiting developmental angiogenesis of the ISV. In agreement, the pik3r1 gene, encoding the p85 regulatory subunit, is expressed at all developmental stages from 4 hours post fertilisation (hpf) to 5 days post fertilisation (dpf). Whereas pik3cA, encoding the p110α subunit, is expressed only at later developmental stages (1-5 dpf).
Given that the p110α isoform has been suggested to be selectively required for angiogenesis, our results unexpectedly suggest that mTOR, and not p110α inhibitors, are the most effective PI3K pathway inhibitors of developmental angiogenesis in zebrafish. In addition, the lack of expression of the p110α isoform at early time-points in zebrafish may account for the differential effects of some inhibitors on ISV and HV angiogenesis. Further investigations of the PI3K pathway and drug combinations hold great promise for the identification of better therapeutics for ocular disease.
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