title: Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity.
abstract: OBJECTIVE
  To determine whether knockdown of Müller cell-derived VEGFA-splice variant, VEGF164, which is upregulated in the rat retinopathy of prematurity (ROP) model, safely inhibits intravitreal neovascularization (IVNV).
  
  
  METHODS
  Short hairpin RNAs for VEGF164 (VEGF164.shRNAs) or luciferase.shRNA control were cloned into lentivectors with CD44 promoters that specifically target Müller cells. Knockdown efficiency, off-target effects, and specificity were tested in HEK reporter cell lines that expressed green fluorescent protein (GFP)-tagged VEGF164 or VEGF120 with flow cytometry or in rat Müller cells (rMC-1) by real-time PCR. In the rat oxygen-induced retinopathy (OIR) ROP model, pups received 1 μL subretinal lentivector-driven luciferase.shRNA, VEGFA.shRNA, or VEGF164.shRNA at postnatal day 8 (P8). Analyses at P18 and P25 included: IVNV and avascular retina (AVA); retinal and serum VEGF (ELISA); density of phosphorylated VEGFR2 (p-VEGFR2) in lectin-labeled retinal endothelial cells (ECs; immunohistochemistry); TUNEL staining and thickness of inner nuclear (INL) and outer nuclear layers (ONL) in retinal cryosections; and pup weight gain.
  
  
  RESULTS
  In HEK reporter and in rMC-1 cells and in comparison to lucifferase.shRNA, VEGFA.shRNA reduced both VEGF120 and VEGF164, but VEGF164.shRNA only reduced VEGF164 and not VEGF120. Compared with luciferase.shRNA, VEGFA.shRNA and VEGF164.shRNA reduced retinal VEGF and IVNV without affecting AVA at P18 and P25. At P25, VEGF164.shRNA more effectively maintained IVNV inhibition than VEGFA.shRNA. VEGFA.shRNA and VEGF164.shRNA reduced pVEGFR2 in retinal ECs at P18, but VEGFA.shRNA increased it at P25. VEGFA.shRNA increased TUNEL+ cells at P18 and decreased ONL thickness at P18 and P25. VEGFA.shRNA and VEGF164.shRNA did not affect pup weight gain and serum VEGF.
  
  
  CONCLUSIONS
  Short hairpin RNA to Müller cell VEGF164 maintained long-term inhibition of IVNV and limited cell death compared with shRNA to VEGFA.
authors.affiliation: Department of Ophthalmology, The John Moran Eye Center, University of Utah, Salt Lake City, Utah.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Jiang
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: David
authors.initials: D
authors.lastname: Culp
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Yang
authors.forename: Lori
authors.initials: L
authors.lastname: Fotheringham
authors.forename: John
authors.initials: J
authors.lastname: Flannery
authors.forename: Scott
authors.initials: S
authors.lastname: Hammond
authors.forename: Tal
authors.initials: T
authors.lastname: Kafri
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Hartnett
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D063928:Ependymoglial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D056965:Injections, Intraocular; D058990:Molecular Targeted Therapy; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015861:Retinal Neovascularization / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D012178:Retinopathy of Prematurity / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D049452:Green Fluorescent Proteins
keywords: Müller cells; intravitreal neovascularization; lentivector; short hairpin RNA; vascular endothelial growth factor