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zhihong yang, Haibo Wang, Yanchao Jiang, Manabu McCloskey, M Elizabeth Hartnett; The combined effect with EPO and VEGF in retinal angiogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5566.
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© ARVO (1962-2015); The Authors (2016-present)
Erythropoietin (EPO) improves cognitive development and replenishes lost red blood cells in preterm infants, but some studies report an increased risk of severe retinopathy of prematurity (ROP) with EPO use. We investigate the role of EPO and its interaction with vascular endothelial growth factor (VEGF) in angiogenesis and address the hypothesis that a neuroprotective form of EPO (carbamylated EPO, cEPO) reduces intravitreal neovascularization (VitNV) in a rat model of oxygen-induced retinopathy (OIR).
Within 4 hours of birth, pups and dams were placed into a controlled oxygen environment where oxygen cycled between 50% and 10% every 24 hours for 14 days. At postnatal day (p)2, p4, and p6, pups received intraperitoneal injections of EPO, cEPO or PBS control. At p18, following 4 days in room air, pups were euthanized and percent avascular retinal area and VitNV were determined from ADPase stained retinal flat mounts. Retinal mRNAs for EPO, EPOR and beta-common receptor were measured by real time PCR. Human retinal microvascular endothelial cell (EC) proliferation following stimulation with EPO, VEGF or control was measured by MTT assay. Phosphorylation and expression of proteins were analysed by Western Blot.
In the rat OIR model, pups injected with cEPO had more VitNV at p18 compared to PBS- or EPO-injected (p<0.05 cEPO vs. EPO; p<0.01 cEPO vs. PBS). VEGF, EPO and EPOR, but not the beta common receptor, were increased in the rat OIR model group compared with room air raised pups at p18. EC proliferation was increased 1.5 fold by EPO or 2 fold by VEGF and increased up to 2.5 fold by their combination. Both EPO and VEGF induced VEGFR2 phosphorylation and downstream phosphorylation of STAT3, AKT, but not ERK, in ECs. EPOR expression level was synergistically increased by EPO and VEGF in ECs. STAT3 inhibitor AG490 reduced EPO-VEGF-induced EC proliferation, but only partially inhibited proliferation induced by their combined stimulation.
Contrary to predicted, cEPO increased VitNV compared to EPO or PBS treated eyes. EPO, EPOR, VEGF and VEGFR2 were increased at p18, whereas the beta common was not, and this may account for the outcomes seen. EPO and VEGF induced EC proliferation and kinase phosphorylation levels. Determining the mechanisms VEGF and EPO interactions may lead to a safe method to improve cognitive development in preterm infants without worsening VitNV.
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