Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity

Abstract

Purpose: 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.

Keywords: Müller cells; intravitreal neovascularization; lentivector; short hairpin RNA; vascular endothelial growth factor.

Figure 1

Generation of lentivector-delivered shRNA for specific knockdown of VEGF164 in Müller cells. HEK reporter cell lines expressed GFP-tagged VEGF₁₂₀ or VEGF₁₆₄ were transfected with RFP-expressed lentivector VEGF164.shRNA plasmids or empty vector without shRNA. (A) Flow cytometry (fluorescence activated cell sorting [FACS]) of GFP fluorescence. (B) Quantification of percent silencing of VEGF₁₂₀ and VEGF₁₆₄ by VEGF164 shRNAs from FACS analysis. (C) Real-time PCR of mRNA of VEGF₁₂₀ and VEGF₁₆₄ in rMC-1 infected without lentivirus (uninfected) or with lentivector-driven shRNA to luciferase (luc.shRNA), VEGFA (VEGFA.shRNA), or VEGF₁₆₄ (VEGF164.shRNA). *P < 0.05 and **P < 0.01 versus luc.shRNA).

Figure 2

In vivo analysis of lentivector-delivered shRNA transduction in retina of pups raised in the rat ROP model at P18 and P25 following subretinal injection at P8. (A) Micron III images show GFP expression in retina of pups following lentivirus injection at P25. (B) GFP expression is localized with CRALBP-labeled Müller cells in retinal cryosections at P25. (C) ELISA of retinal VEGFA protein at P18 and P25. *P < 0.05. **P < 0.001 versus luc.shRNA at P18. †P < 0.05. ††P < 0.01 versus luc.shRNA at P25.

Figure 3

Lentivector-derived shRNA to VEGFA or VEGF₁₆₄ reduces IVNV without affecting physiological retinal vascular development (AVA) in the rat ROP model. Images of retinal flatmounts at P18 and P25 following subretinal injections in each group. (A) luc.shRNA, VEGFA.shRNA, and VEGF164.shRNA. *Points to avascular retinal area. A white arrow points to IVNV. (B) Quantification of IVNV. *P < 0.05. ***P < 0.001 versus luc.shRNA at P8. †P < 0.01 versus VEGFA.shRNA at P25. ###P < 0.001 versus VEGFA.shRNA at P18. (C) Avascular retina.

Figure 4

Analysis of VEGFR2 activation in pups treated with subretinal injections of lentivector-driven shRNAs in the rat ROP model. (A) Immunohistochemistry of p-VEGFR2 in retinal cryosections. (B) Semiquantification of p-VEGFR2 (blue) in total retina (***P < 0.001 versus luc.shRNA at P25). (C) Colabeling of p-VEGFR2 (blue) in lectin (red)-labeled ECs in the primary plexus (depicted within boxes; *P < 0.05, **P < 0.01 versus luc.shRNA at P18; ††P < 0.01 versus luc.shRNA at P25) from P18 and P25 pups treated with luc.shRNA, VEGFA.shRNA, and VEGF164.shRNA.

Figure 5

Analysis of retinal apoptosis and retinal morphological changes in the pups treated with lentivector-driven shRNAs in the rat ROP model. Images of TUNEL staining (A) and number of TUNEL (red) positive cells (B) in retinal DAPI (blue) stained cryosections from P18 and P25 pups treated with luc.shRNA, VEGFA.shRNA, and VEGF164.shRNA (**P < 0.01, ***P < 0.001 versus PBS at P18; †††P < 0.001 versus luc.shRNA at P18). Quantification of the thickness of the INL (C) (***P < 0.001 versus PBS at P18; ††P < 0.01 versus luc.shRNA at P25) and the ONL (D) (***P < 0.001 versus luc.shRNA at P18; ††P < 0.001 versus luc.shRNA at P25) in DAPI-stained retinal cryosections from P18 and P25 OIR pups treated with luciferase.shRNA, VEGFA.shRNA, and VEGF164.shRNA.

Figure 6

Lentivector-driven shRNAs treatment has no effect on pup growth and serum VEGF. Pup weight gains from P8 to P18 or P25 (A) and ELISA of serum VEGF in P18 or P25 pups (B) treated with luc.shRNA, VEGFA.shRNA, and VEGF164.shRNA in the rat ROP model.