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Deeksha Gambhir, Yanchao Jiang, George W Smith, Zhihong Yang, Haibo Wang, Lori Fotheringham, M Elizabeth Hartnett; Mueller cell derived VEGF164 knockdown reduces retinal neovascularisation in the rat model of retinopathy of prematurity.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5388.
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© ARVO (1962-2015); The Authors (2016-present)
ROP primarily affects preterm infants. In ROP and its models, VEGF causes intravitreal neovascularization (IVNV). We found that inhibiting Mueller cell VEGFA reduced IVNV in the rat ROP model. VEGFA is vital for Mueller cell survival, as these nourish other cells in the retina. VEGF164 is a prevalent isoform of VEGF involved in ROP models. We asked whether inhibition of Mueller cell VEGF164 would reduce IVNV and AVA safely in Mueller and other retinal cells in the rat ROP model.
Lentivectors with the CD44 promoter and a short hairpin RNA (shRNA) were used to knockdown VEGFA (shA) or VEGF164 (sh164) in rat Mueller cells. To test the specificity of sh164, r-MC1 cells were infected with control shRNA (shC), shA and sh164 or left uninfected. Sprague-Dawley rat pups were placed in an oxycycler chamber that cycled oxygen between 50% and 10% every 24 hours for 14 days. At p8, pups were administered subretinal injections of shC, shA or sh164. PBS was injected in some eyes to test the effect of lentivirus alone. Pups were moved to room air at p14 and euthanized at p18 or p25 for analyses of mRNA, protein, retinal flat mounts and immunohistochemistry. mRNA levels of VEGF164 and VEGF120 were measured by qPCR. VEGF was measured in retinal lysates by ELISA. % IVNV and avascular retina area (AVA) to total retinal area were determined by retinal flat mounts. TUNEL assay was done on retinal cryosections.
sh164 reduced the mRNA transcript level of VEGF164 (p<0.001), and shA vectors reduced the expression of VEGF164 (p<0.001) and VEGF120 (p<0.05) in r-MC1 cells versus shC or uninfected control. Compared to control shC, retinal VEGF protein levels were significantly reduced by shA at p18 and p25, but at p25 by sh164. IVNV was significantly reduced in shA (80%) and sh164 (50%) at p18.Significant decrease in IVNV by sh164 (45%) was seen at p25. shA or sh164 did not affect AVA. Compared to PBS injected eyes, shC and shA, but not sh164, eyes had high TUNEL positive (+) cells at p18. All groups had low (+) cells at p25.Only shA reduced thickness of the ONL at p18 and p25.
In the rat model of ROP, knockdown of Mueller cell VEGF164 may more safely inhibit IVNV and sustain the effect. Knockdown of Mueller cell VEGFA may reduce cell survival in the INL and ONL in the rat ROP model. Additional studies validating the efficacy and safety of VEGF isoforms is warranted.
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