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Md Imam Uddin, Tyler C. Kilburn, John S Penn; Imaging Endoglin mRNA in Mouse OIR with Anti-Sense Hairpin-DNA Gold-Nanoparticle Conjugates. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5838. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the capacity of novel anti-sense hairpin-DNA gold-nanoparticle conjugates (AS-ENG hAuNP) to image endoglin mRNA in hypoxic mouse retinal microvascular endothelial cells (MRMEC) and in mouse oxygen-induced retinopathy (OIR).
AS-ENG hAuNP and non-sense (NS)-ENG hAuNP were designed and synthesized by conjugating hairpin-DNA oligonucleotides (oligos) incorporating an anti-sense sequence complimentary to endoglin mRNA and that same sequence scrambled. These oligos were modified with a fluorophore reporter and conjugated to gold nanoparticles. Endoglin mRNA expression was induced in MRMECs by exposing them to hypoxia (0.1%O2, 5%CO2) in culture medium containing 2% FBS. Mouse OIR was performed according to a standard protocol. AS-ENG hAuNP and NS-ENG hAuNP were added to the culture medium of hypoxic or normoxic MRMECs, and OIR mice received intraperitoneal injections of these agents. Imaging of endoglin mRNA in MRMEC and in mouse OIR retina was performed using fluorescence microscopy. Fluorescence in situ hybridization was used as an alternative method to validate endoglin mRNA expression in mouse OIR retinas.
Enhancement of AS-ENG hAuNP-dependent fluorescence was clearly visualized in hypoxic MRMEC and in the OIR retina, demonstrating a high signal to noise ratio. As expected, NS-ENG hAuNP yielded no response in either setting. The fluorescence spatially overlapped with pathologic neovascular tufts in retinas from OIR mice, indicating abundant endoglin mRNA expression in these structures. hAuNP were not toxic to MRMECs as assessed by a live-dead assay. Fluorescence in situ hybridization confirmed the same endoglin mRNA expression patterns as those obtained using hAuNP. To the best of our knowledge, this is the first report demonstrating the application of AS-ENG hAuNP for imaging endoglin mRNA in pathologic retinal tissues.
This study demonstrates the utility of AS-hAuNP, a new hybrid nanotechnology to image endoglin mRNA in mouse OIR. We propose that this method can be generally extended to image other mRNAs associated with ocular pathologies, allowing its application to predict morbidity, monitor disease progression and/or therapeutic response.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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