Abstract
Oxygen-induced retinopathy (OIR) upregulates Müller cell vascular endothelial growth factor A (VEGFA) that causes intravitreal neovascularization similar to severe retinopathy of prematurity (ROP). Safety concerns exist with anti-VEGF treatment for ROP. We evaluated long-term knockdown of Müller cell-VEGFA with short-hairpin RNAs to VEGFA or VEGF164 via subretinal lentivirus delivery (L-VEGFAshRNA, L-VEGF164shRNA) on retinal structure and function in a rat OIR model. Lectin-stained retinal flat mounts analyzed for areas of avascular/total retina (AVA) and intravitreal neovascular/total retina (IVNV) showed initial significantly reduced IVNV by L-VEGFAshRNA and L-VEGF164shRNA compared to control, luciferase-shRNA lentivirus, without late recurrence. Spectral-domain optical coherence tomography (OCT) and immunohistochemical sections (IHC) demonstrated changes in retinal layer thicknesses in L-VEGFAshRNA or L-VEGF164shRNA compared to control. Ganzfeld electroretinograms were increased in L-VEGFAshRNA or L-VEGF164shRNA compared to control. Erythropoietin (EPO), brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3) mRNAs were increased in L-VEGFAshRNA, but not L-VEGF164shRNA retinas. In cultured rat Müller cells, knockdown of VEGF upregulated NT-3 and EPO, whereas treatment with EPO activated neuroprotective signaling. Methods to reduce IVNV by selective knockdown of VEGFA, and particularly VEGF164, in Müller cells may have fewer deleterious effects than nonselective VEGFA inhibition to all cells in the retina.
Highlights
Retinopathy of prematurity (ROP) is a leading cause of childhood vision loss and blindness worldwide[1] and is increasing with survival of extremely preterm infants[2]
Lentiviruses were used to deliver vectors containing the cell specific CD44 promoter to drive the expression of short hairpin RNAs (shRNAs) targeting vascular endothelial growth factor A (VEGFA) (L-VEGFAshRNA), VEGF164 (L-VEGF164shRNA) or luciferase (L-lucifshRNA) as a non-mammalian control within Müller cells
Efficiency was previously confirmed in HEK 293 green fluorescent protein (GFP) reporter cell lines expressing either rat VEGF120 to assess VEGFA knockdown or VEGF164 to assess the VEGF164 splice variant
Summary
Retinopathy of prematurity (ROP) is a leading cause of childhood vision loss and blindness worldwide[1] and is increasing with survival of extremely preterm infants[2]. When the infant is moved from supplemental oxygen to room air, poor oxygenation of the avascular retina stimulates disordered growth of retinal blood vessels into the vitreous rather than into the avascular retina This intravitreal neovascularization can develop into severe, vision-threatening ROP3. Studies in animal models representative of severe ROP in humans show that inhibition of VEGF with neutralizing intravitreal antibodies at certain doses effective at inhibiting retinopathy reduce pup growth, reduce retinal capillary density and result in recurrent intravitreal neovascularization in association with activation of angiogenic signaling pathways in the retina[11,12]. Selective knockdown of VEGF164 in Müller cells with a lentivirus carrying VEGF164 shRNA driven by a cell specific promoter resulted in reduced intravitreal neovascularization in a rat oxygen-induced retinopathy (OIR) model[14] without thinning of the outer nuclear layer in the short-term. We determined whether long-term inhibition of overexpressed VEGF164 in Müller cells would be sufficient to inhibit intravitreal neovascularization without causing functional or structural loss to the retina and to explore effects of targeted VEGF knockdown in Müller cells on the retina
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