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Manabu McCloskey, Yanchao Jiang, Haibo Wang, zhihong yang, Jeremy Strange, Tal Kafri, John Flannery, M Elizabeth Hartnett; Knockdown of Mueller cell specific VEGF reduces retinal neovascularization in a rat model of retinopathy of prematurity (ROP). Invest. Ophthalmol. Vis. Sci. 2013;54(15):621.
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Increased VEGF causes intravitreal neovascularization (IVNV) in a rat ROP model, but inhibition of VEGF may have adverse effects on the developing retina in preterm infants. We hypothesized that inhibition of cell-specific expression of VEGF may be a safe way to inhibit IVNV.
Newborn rat pups were exposed to repeated fluctuations in oxygen between 50% and 10% inspired oxygen in a controlled environment (Oxycycler, Biospherix) every 24 hours for 14 days (rat OIR model). Short hairpin RNAs to VEGF or control luciferase fashioned as microRNAs with green fluorescent protein tags were cloned into a lentiviral transfer vector with a CD44 promoter to specifically target Mueller cells. At postnatal day (p) 8, the pups received subretinal injections of 1ul of shRNA-VEGF or shRNA-luc. At p18, pups were weighed and eyes were processed for analysis of in situ hybridization for mRNA localization, protein for ELISA, percent avascular area (AVA) and intravitreal neovascularization (IVNV) of total retinal areas from lectin stained flat mounts, and sections for immunohistochemistry.
At p14, VEGF localized to retinal layers corresponding to retinaldehyde-binding protein-stained Mueller cells. Increased VEGF protein was detected in pups from the rat OIR model compared to room air raised pups (p < 0.001). IVNV was reduced 75% by VEGFA-shRNA (p < 0.001) compared to Lucif-sh. AVA and body weights were unaffected compared to control. Retinal VEGF protein was reduced in shRNA-VEGF (p <0.05) compared to Lucif-sh (p < 0.01), and shRNA-VEGF reduced VEGF level to room air.
In the rat model of ROP, knockdown of Mueller cell-derived VEGF significantly reduced IVNV compared to Luci-sh control and PBS injected. shRNA-VEGF was able to reduce retinal VEGF protein level to that of p18 room air raised pups. Mueller cell derived VEGF is a significant factor leading to IVNV in the rat model of ROP. Further study into cell and molecular mechanisms driving pathological IVNV and AVA is warranted.
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