VEGF/VEGFR2 blockade does not cause retinal atrophy in AMD-relevant models
Da Long, Yogita Kanan, Jikui Shen, Sean F. Hackett, Yuanyuan Liu, Zibran Hafiz, Mahmood Khan, Lili Lu, Peter A. Campochiaro
Da Long, Yogita Kanan, Jikui Shen, Sean F. Hackett, Yuanyuan Liu, Zibran Hafiz, Mahmood Khan, Lili Lu, Peter A. Campochiaro
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Research Article Angiogenesis Ophthalmology

VEGF/VEGFR2 blockade does not cause retinal atrophy in AMD-relevant models

Abstract

Intraocular injections of VEGF-neutralizing proteins provide tremendous benefits in patients with choroidal neovascularization (NV) due to age-related macular degeneration (AMD), but during treatment some patients develop retinal atrophy. Suggesting that VEGF is a survival factor for retinal neurons, a clinical trial group attributed retinal atrophy to VEGF suppression and cautioned against frequent anti-VEGF injections. This recommendation may contribute to poor outcomes in clinical practice from insufficient treatment. Patients with type 3 choroidal NV have particularly high risk of retinal atrophy, an unexplained observation. Herein we show in mouse models that VEGF signaling does not contribute to photoreceptor survival and functioning: (a) neutralization of VEGFR2 strongly suppresses choroidal NV without compromising photoreceptor function or survival; (b) VEGF does not slow loss of photoreceptor function or death in mice with inherited retinal degeneration, and there is no exacerbation by VEGF suppression; and (c) mice with type 3 choroidal NV develop retinal atrophy due to oxidative damage with no contribution from VEGF suppression. Intraocular injections of VEGF-neutralizing proteins, a highly effective treatment in patients with neovascular AMD, should not be withheld or reduced due to concern that they may contribute to long-term visual loss from retinal atrophy.

Authors

Da Long, Yogita Kanan, Jikui Shen, Sean F. Hackett, Yuanyuan Liu, Zibran Hafiz, Mahmood Khan, Lili Lu, Peter A. Campochiaro

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Figure 3

Type 3 choroidal neovascularization (NV) causes photoreceptor cell death from oxidative damage.

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Type 3 choroidal neovascularization (NV) causes photoreceptor cell death...
Rho/VEGF mice with type 3 choroidal NV were given normal drinking water or drinking water containing 7 mg/ml N-acetylcysteine (NAC) and compared with wild-type mice. At postnatal day 21 (P21), P28, and P56, mice from each of these 3 groups were euthanized and frozen ocular sections were immunohistochemically stained for 4-hydroxynonenal (HNE, A) or 3-nitrotyrosine (3-NT, B) and vascular cells were stained with FITC-labeled Griffonia simplicifolia agglutinin (GSA) lectin. Nuclei were stained with Hoechst. (A) At P21, untreated rho/VEGF mice (control) showed HNE (red, arrows) along the outer border of the outer nuclear layer (ONL) and in the subretinal space adjacent to GSA-stained NV (green, asterisks), which was increased at P28 and P56 when there was also staining within the ONL and inner nuclear layer (INL). In NAC-treated rho/VEGF mice there was minimal HNE at each of the time points but NV (green, asterisks) was seen within the ONL and subretinal space. At P28, some mild staining for HNE was seen adjacent to and within NV in the subretinal space. There was no HNE or NV seen in wild-type mice. (B) Control rho/VEGF mice also showed 3-NT along the outer border of the ONL (red, arrows) adjacent to NV (green, asterisks) at all 3 time points with some staining in the INL at the later time points, whereas there was little 3-NT in NAC-treated rho/VEGF mice, and none in wild-type mice. (C) Retinal flat mounts stained with peanut agglutinin (red) and GSA (green) showed evenly spaced cone matrix sheaths and no NV in P56 wild-type mice (left panel), whereas P56 rho/VEGF mice (right panel) showed dropout of cone matrix sheaths adjacent to NV (arrows). (D) ELISA of retinal homogenates showed a significant reduction in mean (±SEM) protein carbonyl content in NAC-treated rho/VEGF mice compared with control rho/VEGF mice (n = 6 for each group). **P < 0.01 by unpaired t test. (E) Hematoxylin-stained retinal sections from the same location in the retina (I3) show an ONL with irregular borders in untreated (control) and NAC-treated P56 rho/VEGF mice (NAC), but the ONL appeared thicker in NAC-treated mice and more comparable to the ONL in P56 wild-type mice. Image analysis by an investigator masked with regard to treatment group showed that mean (±SEM) ONL thickness was significantly reduced in control versus NAC-treated rho/VEGF mice at 4 of 6 measurement locations in the vertical meridian (n = 6 for each group). *P < 0.05, **P < 0.01 by unpaired t test. For context, mean (±SEM) ONL thickness at the same 6 locations is shown for P56 wild-type mice (n = 6). (F) A representative immunoblot for rhodopsin kinase (GRK1) shows a stronger signal in a retinal homogenate from a P56 NAC-treated rho/VEGF mouse compared with an untreated P56 rho/VEGF mouse (control). Densitometry showed that the mean (±SEM) GRK1/actin ratio was significantly greater in NAC-treated mice (n = 4). *P < 0.05 by unpaired t test.