Dataset Information

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

ABSTRACT: 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).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 ?L 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.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.Short hairpin RNA to Müller cell VEGF164 maintained long-term inhibition of IVNV and limited cell death compared with shRNA to VEGFA.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC3920823 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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

Jiang Yanchao Y Wang Haibo H Culp David D Yang Zhihong Z Fotheringham Lori L Flannery John J Hammond Scott S Kafri Tal T Hartnett M Elizabeth ME

Investigative ophthalmology & visual science 20140210 2

<h4>Purpose</h4>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).<h4>Methods</h4>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 ...[more]

PMID: 24425851

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Project description:BackgroundRetinopathy of prematurity is a leading cause of childhood blindness worldwide. Peripheral retinal ablation with conventional (confluent) laser therapy is destructive, causes complications, and does not prevent all vision loss, especially in cases of retinopathy of prematurity affecting zone I of the eye. Case series in which patients were treated with vascular endothelial growth factor inhibitors suggest that these agents may be useful in treating retinopathy of prematurity.MethodsWe conducted a prospective, controlled, randomized, stratified, multicenter trial to assess intravitreal bevacizumab monotherapy for zone I or zone II posterior stage 3+ (i.e., stage 3 with plus disease) retinopathy of prematurity. Infants were randomly assigned to receive intravitreal bevacizumab (0.625 mg in 0.025 ml of solution) or conventional laser therapy, bilaterally. The primary ocular outcome was recurrence of retinopathy of prematurity in one or both eyes requiring retreatment before 54 weeks' postmenstrual age.ResultsWe enrolled 150 infants (total sample of 300 eyes); 143 infants survived to 54 weeks' postmenstrual age, and the 7 infants who died were not included in the primary-outcome analyses. Retinopathy of prematurity recurred in 4 infants in the bevacizumab group (6 of 140 eyes [4%]) and 19 infants in the laser-therapy group (32 of 146 eyes [22%], P=0.002). A significant treatment effect was found for zone I retinopathy of prematurity (P=0.003) but not for zone II disease (P=0.27).ConclusionsIntravitreal bevacizumab monotherapy, as compared with conventional laser therapy, in infants with stage 3+ retinopathy of prematurity showed a significant benefit for zone I but not zone II disease. Development of peripheral retinal vessels continued after treatment with intravitreal bevacizumab, but conventional laser therapy led to permanent destruction of the peripheral retina. This trial was too small to assess safety. (Funded by Research to Prevent Blindness and others; ClinicalTrials.gov number, NCT00622726.).

Project description:To determine incidence, risk factors, risk period, and characteristics of recurrent retinopathy of prematurity (ROP) treated by intravitreal bevacizumab (IVB) monotherapy.Retrospective case series.Premature infants with type 1 ROP (subdivided into stage 3+ ROP and aggressive posterior ROP [APROP]) in zone I or zone II posterior who received IVB monotherapy and were followed up for at least 65 weeks adjusted age (AA).Retrospective review of infants who demonstrated recurrence of type 1 ROP after IVB monotherapy, including examination of RetCam fundus photographs and fluorescein angiograms.Incidence, risk factors, risk period, and characteristics of recurrent ROP.Intravitreal bevacizumab monotherapy in 241 infants (471 eyes) was reviewed. Recurrence incidence was 8.3% (20/241) for infants and 7.2% (34/471) for eyes. Recurrence risk factors of greatest significance were appearance of neovascularization as APROP (P = 0.006), extended duration of hospitalization (P = 0.01), and lower birth weight (P = 0.024). Recurrence risk period was between approximately 45 and 55 weeks AA (90.0% [18/20] for infants and 94.1% [32/34] for eyes), with mean recurrence of 51.2 weeks AA (±4.6 weeks; range, 45.7-64.9 weeks) and mean interval of 16.2 weeks (±4.4 weeks) between treatments. Recurrence characteristics included plus disease (20/20 infants [100%]) and neovascularization, which appeared at the following sites: stage 3+ ROP with confluent neovascularization recurred both at the advancing edge and at the initial ridge and extraretinal fibrovascular proliferative complex (12/14 infants [85.7%]). However, APROP (6/6 infants [100%]) and stage 3+ ROP with nonconfluent neovascularization (2/14 infants [14.3%]) recurred only at the advancing edge. Also, the anterior extent of retinal vascularization was decreased (mean, 1.76 disc diameters [DD] vs. 4.48 DD), and the rate of retinal vascularization was delayed (mean, 0.11 DD/week vs. 0.23 DD/week) in those with versus without recurrence, respectively. After retreatment with IVB, retinal vascularization proceeded minimally and slowly.Premature children with severe ROP are being treated successfully with IVB monotherapy. However, recurrence is not uncommon, so vigilant follow-up is necessary to ensure timely re-treatment. Knowledge of recurrence incidence, risk factors, risk period, and characteristics allows for tailored clinical management.

Project description:OBJECTIVE:To determine the effects of oxygen fluctuations on pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF)/PEDF ratios in a relevant rat model of retinopathy of prematurity (ROP). METHODS:The expression of retinal PEDF mRNA and of VEGF and PEDF protein were determined using real-time polymerase chain reaction or enzyme-linked immunosorbent assays at different postnatal day ages for rat pups raised in room air (RA) or in a rat model mimicking ROP. Statistical outcomes were determined with factorial analyses of variance. Mean VEGF and PEDF protein levels were determined at different ages for rats in the ROP model and for RA-raised rats, and the ratio of VEGF/PEDF protein versus age was plotted. At postnatal day (P) 14, inner retinal plexus vascularization had extended to the ora serrata in pups raised in RA. In the ROP model, avascular retina persisted at P14 and intravitreous neovascularization developed at P18. Therefore, VEGF and PEDF expression was determined in the ROP model and in RA-raised rat pups at P14 and P18. RESULTS:Older age was associated with increased PEDF mRNA (p<0.001), PEDF protein (p=0.005), and VEGF protein (p=0.005), and VEGF protein (p<0.0001). Exposure to fluctuations of oxygen in the 50/10 oxygen-induced retinopathy model compared to RA was associated with increased PEDF mRNA (p=0.0185), PEDF protein (p<0.0001), or VEGF protein (p<0.0001). The VEGF/PEDF ratio favored angiogenic inhibition (<1.0) before but not on P14, when avascular retina persisted in the ROP model but not in RA. The VEGF/PEDF ratio favored angiogenesis (>1.0) at P14 and P 18 when intravitreous neovascularization occurred in the ROP model. CONCLUSIONS:Increased expression levels of VEGF and PEDF are associated with older postnatal day age or with exposure to fluctuations in oxygen in the 50/10 oxygen-induced retinopathy model compared to RA. PEDF protein more closely associates with avascular retinal features and neovascularization than does VEGF protein or the VEGF/PEDF in the ROP model. Although PEDF has been proposed as a potential treatment in ROP, interventional studies using PEDF in an ROP model to potentially reduce intravitreous neovascularization are required to determine timing, efficacy, and dose of PEDF.

Dataset Information

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

Publications

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

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