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Aaron B Simmons, Eric Kunz, Haibo Wang, Colin Andrew Bretz, Thipparat Suwanmanee, Tal Kafri, M Elizabeth Hartnett; Knockdown of endothelial cell VEGFR2 reduces intravitreal neovascularization in the rat model of Retinopathy of Prematurity. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5469.
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The use of intravitreal agents that broadly inhibit the bioactivity of vascular endothelial growth factor (VEGF) effectively inhibit intravitreal neovascularization (IVNV) in models of oxygen-induced retinopathy (OIR), but do not permit complete physiological retinal vascular development (PRVD), raising concern about their translation to human Retinopathy of Prematurity (ROP). The purpose of this study was to address the hypothesis that knockdown of VEGF receptor 2 (VEGFR2) specifically in endothelial cells (ECs) would reduce IVNV without adversely affecting PRVD, by using an OIR model representative of ROP.
Lentiviruses were designed to deliver plasmids in which the VE-cadherin promoter was used to drive expression of mircoRNA30 embedded short hairpin RNAs (shRNAs) to VEGFR2 and a GFP tag (L-VEGFR2shRNA). A control plasmid with shRNA to the non-mammalian gene, luciferase was also developed (L-LUCshRNA). Transduction efficiency of L-VEGFR2shRNA was assessed by GFP expression in transduced rat retinal microvascular ECs (rRMVECs) compared to uninfected rRMVECs and for knockdown efficiency by real-time PCR compared to L-LUCshRNA transduced or uninfected rRMVECs. The 50/10 rat OIR model was used: newborn Sprague Dawley rat pups were placed into a controlled oxygen environment (Oxycycler, Biospherix) within 6 hours of birth that cycled oxygen between 50% and 10% every 24 hours for 14 days. At postnatal day (p) 8, pups received bilateral 1 microliter injections of L-VEGFR2shRNA, L-LUCshRNA or PBS. At p14, pups were moved to room air and euthanized at p20. Retinal flatmounts were stained with the EC marker isolectin GS-IB4 (lectin), imaged and areas of avascular/total retina (AVA) and intravitreal neovascularization/total retina (IVNV) were measured. The specificity of L-VEGFR2shRNA in-vivo was assessed by co-staining with GFP and lectin in retinal flatmounts and cryosections.
L-VEGFR2shRNA transduced rRMVECs and reduced VEGFR2 mRNA levels compared to L-LUCshRNA (p= 0.04) or uninfected control (p= 0.004). L-VEGFR2shRNA transduction was localized to ECs in-vivo. IVNV was significantly reduced in L-VEGFR2shRNA compared to L-LUCshRNA (1.9% vs. 1.2%, 37% reduction, p=0.02, n≥13), as was AVA (26% vs. 37%, 30% reduction, p=0.02, n≥13).
Knockdown of VEGFR2 specifically in ECs reduced IVNV without increasing AVA in a model representative of ROP.
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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