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Alena Bartakova, Geoffrey Weiner, Michael Oldham, Napoleone Ferrara, Richard Daneman, Eric Nudleman; Endothelial cell specific gene expression changes in Oxygen Induced Retinopathy (OIR). Invest. Ophthalmol. Vis. Sci. 2018;59(9):5470. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinopathy of prematurity (ROP) is an important cause of blindness in premature children worldwide. Visual morbidity in ROP is a consequence of incomplete retinal vascularization, resulting in ischemia and compensatory neovascularization. In addition, ROP results in a breakdown of the blood-retinal barrier (BRB). Although these changes in the retinal vascular endothelial cells (RVECs) are central to the disease phenotype, little is known about the underlying molecular changes. Using the mouse OIR model, we aimed to identify the endothelial cell specific transcriptional changes in purified RVECs.
To induce ROP, we exposed C57/Bl6 mice to 75% oxygen from postnatal day 7 (P7) until P12, and then returned to room air to induce relative hypoxia. OIR animals and room air controls were sacrificed at P12, P17 and P25. To isolate RVECs from mice, the retinas were dissected and enzymatically and mechanically dissociated to recover a single cell suspension. RVECs were purified by immunopanning using an adapted protocol (Zhou, Sohet, and Daneman 2013). Contaminating perictytes, leucocytes and microglia were removed by negative selection; RVECs were then positively selected using a CD31– coated plate. Isolated RVEC mRNA was purified and used for high-throughput RNA sequencing. Eigengene network study was applied to isolate the endothelial cell module from potential contaminants. Results were analyzed comparing gene expression in RVECs between OIR and control samples.
Gene expression analysis shows successful mapping to the mouse genome, and confirms our ability to extract sufficient quantities of high quality mRNA. Moreover, the purity of our cell extraction and immunopanning protocol is supported by the high levels of expression of vascular endothelial-specific genes. The gene expression analysis shows major alterations between OIR and room air animals, providing novel targets that regulate BRB integrity and retinal vascular proliferation.
Our results show changes in endothelial cell specific gene expression between OIR and normoxic animals during the vasoobliteration, vasoproliferation, and resolution phases. This data set provides novel genes whose expression may have important functional significance on aberrant vascular proliferation and breakdown of the BRB. Future studies will be aimed at determining the functional roles of these molecules.
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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