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Matthew Hartzell, Andrew Stempel, Trevor McFarland, Binoy Appukuttan, Tim Stout; Assessment of Intravitreal AAV-TEAD4 Isoforms in the OIR Model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2745.
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Purpose: The mouse model of oxygen-induced retinopathy (OIR) has been used extensively as a model of retinopathy of prematurity to study retinal neovascularization (NV). We have shown that the transcription factor TEAD4, and its isoforms, can influence VEGF expression; a determining factor driving tuft formation in this model. The purpose of this study was to observe the effects of TEAD4 isoforms on retinal NV within this model.
Methods: Postnatal day 7 (P7) C57BL/6 mice were injected intravitreally with AAV-DJ containing either the TEAD4 1305 or 447 isoforms (n=4), AAV-DJ containing GFP (n=4) or PBS (n=4). The mice were then introduced to a 75% oxygen environment for five days before recovering in room air. All mice were sacrificed at P17 and eyes were enucleated. One eye from each mouse was enucleated, fixed in 10% neutral buffered formalin and paraffin embedded. Tissue was sectioned at 5uM and sequential slides were stained for hematoxylin and eosin. Neovascular tuft nuclei were manually counted (15 slides/eye). The retina from the contralateral eye was dissected and used for western blot analysis using anti-TEAD4 antibodies to confirm viral introduction of human isoforms.
Results: Western blotting confirmed the presence of the exogenous TEAD4 1305 and 447 isoforms. The AAV 447, 1305 and GFP injected eyes averaged 3.2, 1.6 and 5.1 tuft nuclei per section respectively. PBS injected eyes had an average of 11.75 tuft nuclei per section.
Discussion: Successful ocular transduction of P7 OIR mice was achieved with AAV-DJ viruses encoding the TEAD4 1305 and 447 isoforms. Neovascular tuft nuclei counts suggest no stimulatory effect with the introduction of stimulatory TEAD4 isoforms in this model. The presence of elevated levels of TEAD4 in this model may influence the expression of VEGF, VEGF receptor and/or other angiogenic factors, perhaps leading to the decrease in overall tuft formation in the treated groups.
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