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Aaron B Simmons, Eric Kunz, Colin Andrew Bretz, Haibo Wang, Thipparat Suwanmanee, Tal Kafri, M Elizabeth Hartnett; Knockdown of the erythropoietin receptor in endothelial cells extends physiological retinal vascular development in a model of retinopathy of prematurity. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1656.
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© ARVO (1962-2015); The Authors (2016-present)
Erythropoietin (EPO) signaling through its receptor (EPOR) has been implicated in angiogenesis in clinical and experimental studies. However, it remains unclear how EPOR signaling in endothelial cells (ECs) affects retinal physiologic and pathologic angiogenesis. Here we tested the hypothesis that EPOR signaling in ECs was involved in pathological intravitreal neovascularization but not physiological retinal vascular development (PRVD) in retinopathy of prematurity (ROP) by using the rat oxygen-induced retinopathy (OIR) model representative of ROP.
Lentiviral vectors with the VE-cadherin promoter and a GFP tag were generated to deliver shRNAs to EPOR (EC-shEPOR) or control luciferase (EC-shLUC). EC-shEPOR knockdown efficiency was assessed in rat retinal microvascular ECs (rRMVECs) transduced with either EC-shEPOR or EC-shLUC and analyzed for EPOR mRNA with real time PCR. Rat OIR model: Within 6 hours of birth, Sprague Dawley rat pups with a nursing dam were placed into a chamber that cycled oxygen between 50% and 10% every 24 hours for 14 days. At postnatal day (P) 8, pups received 1 μl bilateral subretinal injections of EC-shEPOR or EC-shLUC. At P14, pups were moved to room air and euthanized at P20. Areas of peripheral avascular/retinal area (AVA; inverse of PRVD) and intravitreal neovascular/retinal area (IVNV) were measured in lectin stained retinal flatmounts. EC-shEPOR specificity was verified by labeling retinal flatmounts and sections with anti-GFP and lectin. Retinal thickness was measured in DAPI stained retinal sections. Data were statistically analyzed using a multiple linear regression (MLR) model that controls for biological diversity between litters (OIR data) or a t-test (mRNA data).
Compared to EC-shLUC, rRMVECs transduced with EC-shEPOR had ~40% reduced EPOR mRNA (t-test, p= 0.009, n=4). GFP localized to ECs in lentiviral-transduced eyes. AVA was significantly reduced in EC-shEPOR treated compared to EC-shLUC treated eyes (31±4% vs. 39±3%, MLR, p=0.048, n=15), but IVNV was not significantly different (2.4±0.4% vs. 2.6±0.3%, MLR, p=0.648, n=15). No differences in retinal thicknesses were detected in EC-shEPOR treated compared to EC-shLUC treated eyes (MLR, p>0.3, n≥9).
Our results identify EPOR signaling within ECs as important for PRVD but not IVNV in a model representative of ROP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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