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Jordan J Toutounchian, Jayaprakash Pagadala, Duane D Miller, Jena J Steinle, Ryan Yates; The role of a Src/FAK-paxillin signalsome in VEGF-induced retinal neovascularization. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):208.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular endothelial growth factor (VEGF) is a well-established promoter and biomarker of pathological neovascularization in diabetic retinopathy (DR) and age-related macular degeneration (AMD). In the present study, we have developed a novel strategy of modulating VEGF-induced activation of the FAK/SRC-paxillin signalsome to prevent neovascular eye disease.
We designed compound JP-153 to target paxillin for selective modulation. Using human primary retinal endothelial cells (RECs) we measured proliferation with the tetrazolium salt, WST-1. We used a scratch wound and transwell migration assay plate to assess REC migration. Immunoblotting was carried out to identify FA signaling in RECs. We loaded JP-153 into a topical ophthalmic microemulsion for administration in the in vivo murine oxygen-induced retinopathy (OIR) model of proliferative retinopathy.
VEGF enhanced REC proliferation and migration over 24 hours with significant activation of FA proteins paxillin and FAK within just 15 minutes. JP-153 (0.5 µM) prevented the activation of FA assembly sites: paxillin Y118 and FAK Y576/577 (P < 0.001) independent of ERK-1/2 (P>0.05), a regulator of FA disassembly. JP-153 inhibited proliferation and migration at concentrations ranging from 0.1 to 0.5 µM (P < 0.001). In the murine OIR model, JP-153 dose-dependently prevented neovascular tufting and increased avascular area (P < 0.001; n = 8-14 mice/group). Immunoblotting analysis of whole-retina homogenates revealed a dose-dependent reduction in total paxillin levels following JP-153 treatment (P < 0.05).
JP-153 afforded sub-micromolar potency to inhibit REC proliferation and migration, in vitro. Additionally, the topical administration of JP-153 was successful in delivering therapeutic doses of JP-153 to the retina. Together, our data highlight the essential and dynamic role of paxillin in regulating pathological RNV. In summary, we have discovered the first small-molecule modulator of paxillin signaling that is capable of disrupting VEGF-induced FAK/Src-paxillin activation, in vitro and in vivo. We thus postulate that paxillin is a potential viable target in treating neovascular disease, the leading cause of blindness in humans.
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