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Andrew Stempel, Catherine Morgans, Tim Stout, Binoy Appukuttan; Detection and Quantification of VEGF, HIF-1α, HIF-2α and TEAD4 Transcripts in the Mouse Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5557.
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© ARVO (1962-2015); The Authors (2016-present)
Expression of VEGF under ischemic conditions is regulated by various transcription factors, including HIF-1α and HIF-2α. Recently, the transcription factor TEAD4 has been shown to increase VEGF and HIF-1α transcripts. In this study we use the novel RNAscope method of in situ hybridization in parallel with quantitative real-time PCR (qRT-PCR) to localize and quantify VEGFA, HIF-1α, HIF-2α and TEAD4 transcripts within the retina in the mouse model of oxygen-induced retinopathy (OIR).
Postnatal day (P)7 C57Bl/6 mice were exposed to 75% oxygen for 5 days and then recovered in room air. OIR and room air control animals were sacrificed at P7, P8, P12, P13, P15, P17 P21 and P25. One eye was fixed for 72 hours, paraffin embedded and sectioned at 5µm. mRNA was detected via in situ hybridization using probes for VEGFA, HIF-1α, HIF-2α and TEAD4 (Advanced Cell Diagnostics) and visualized using the RNAscope 2.0 Red Detection System (ACD). The contralateral retina was dissected, RNA was isolated, and mRNA expression was evaluated using qRT-PCR with primers for VEGFA, HIF-1α, HIF-2α and TEAD4. Cell specificity was confirmed by immunohistochemistry for GS-lectin and GFAP.
VEGF mRNA was observed mostly within Mueller cells as well as the ganglion cell and inner nuclear layer. Expression decreased under hyperoxia and rapidly increased upon the return to normoxia with a peak at P15. TEAD4 mRNA levels mirror that of VEGF by qRT-PCR and were observed sporadically throughout the retina, concentrated within retinal vessels. TEAD4 expression co-localizes with GS-lectin within the neovascular tufts as well as GFAP stained Mueller cells at P17 OIR. HIF-2α expression increases during development and predominantly hybridizes to retinal vessels and neovascular tufts as confirmed by lectin co-localization. HIF-1α is constitutively expressed throughout all layers of the retina.
RNAscope is a novel method of in situ hybridization, a powerful tool for retinal cell research. This method can be modified to confirm cell-specific expression with dual immunohistochemical analysis on the same tissue section. Although, VEGF and HIF-1α transcripts are temporally abundant within the retina, both are notably absent from neovascular tufts. Interestingly, HIF-2α and TEAD4 co-localize within the neovascular tufts at the peak of disease.
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