Targeted Knockdown of Overexpressed VEGFA or VEGF164 in Müller cells maintains retinal function by triggering different signaling mechanisms

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.

Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy

“…The LKC UTAS Visual Diagnostic System with BigShot Ganzfeld was used to record Ganzfeld ERG. The ERG was recorded using white light stimulus as previously described [ 15 ]. ERGs were recorded at p34.…”

“…Based on previous studies [ 15 ], at least 16 flat mounts for AVA and IVNV are analyzed per group. A mixed effects linear regression model was used to statistically analyze data collected from OIR rats using STATA-14 software (StataCorp LLC, College Station, TX) that accounted for biological variation between litters and eyes.…”

“…We developed cell-specific lentiviral gene therapy to knock down the VEGF ligand or a splice variant, VEGF 164 , to retinal levels of a room air raised pup of the same developmental age [ 14 ]. Despite efficacy at IVNV without adversely affecting physiological retinal vascular development, knockdown of Müller cell VEGF resulted in structural changes to the neural retina [ 15 ]. We also found that reduced VEGF secretion by Müller cells activated compensatory mechanisms for cell survival, which may have been a response from neural and glial cells that use VEGF as a survival mechanism in response to stress [ 16 ].…”

Relationship between Retinal Thickness Profiles and Visual Outcomes in Young Adults Born Extremely Preterm

“…Analyzing the parafoveal and perifoveal changes in premature infants could be applicable to rodent models, where a fovea is absent, to explore the pathophysiology and potential therapeutic options for ROP. 29,30 Of note, preterm children are also at an increased risk of developing cortical or cerebral visual impairment (CVI). 31 The CVI is impaired vision without primary ocular pathology and it is one of the leading causes of poor visual acuity among EP M A N U S C R I P T A C C E P T E D births.…”

Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity

“…In the retina, pathologic angiogenesis is associated with Müller cell HIF-2α expression (Weidemann et al, 2010;Becker et al, 2018). Here we show that ablation of HIF-2α does not lessen the effect of HIF stabilization in the prevention of experimental ROP, indicating that HIF-2α stabilization is not required for DMOG or Roxadustat induced protection.…”