Inefficacy of anti-VEGF therapy reflected in VEGF-mediated photoreceptor degeneration

Abstract

Retinal neovascularization (RNV) is primarily driven by vascular endothelial growth factor (VEGF). However, current anti-VEGF therapies are limited by short half-lives and repeated injections, which reduce patient quality of life and increase medical risks. Additionally, not all patients benefit from anti-VEGF monotherapy, and some problems, such as unsatisfactory vision recovery, persist after long-term treatment. In this study, we constructed a recombinant adeno-associated virus (AAV), AAV2-SPLTH, which encodes an anti-VEGF antibody similar to bevacizumab, and assessed its effects in a doxycycline-induced Tet-opsin-VEGFA mouse model of RNV. AAV2-SPLTH effectively inhibited retinal leakage, RNV progression, and photoreceptor apoptosis in a Tet-opsin-VEGF mouse model. However, proteomic sequencing showed that AAV2-SPLTH failed to rescue the expression of phototransduction-related genes, which corresponded to reduced photoreceptor cell numbers. This study suggests that anti-VEGF monotherapy can significantly inhibit RNV to some extent but may not be enough to save visual function in the long term.

Keywords: MT: Delivery Strategies; anti-VEGF monotherapy; gene therapy; oxidative stress; photoreceptor degeneration; retinal neovascularization.

Graphical abstract

Figure 1

Construction and in vitro testing of AAV2-SPLTH (A) Schematic representation of AAV2-SPLTH vector. ITR, inverted terminal repeat of AAV2; CBA, chicken beta-actin promoter; T2A, Thosea asigna virus 2A self-cleaving peptide; BGH poly(A), bovine growth hormone polyadenylation signal. (B) Antigen specificity of heavy-chain and light-chain antibodies secreted by AAV2-SPLTH under reducing conditions. (C) Antigen specificity of IgG1 antibodies secreted by AAV2-SPLTH under non-reducing conditions. (D) The anti-VEGF secreted in the supernatants was determined using ELISA. Data are presented as mean ± SD (n = 3).

Figure 2

AAV2-SPLTH inhibits proliferation, migration, and tube formation of HUVECs (A) The results of cell proliferation assay of HUVECs after different treatments. Data are presented as mean ± SD (n = 3). (B) The binary image results of wound-healing assays using HUVECs at 0 and 10 h after different treatments. Scale bars: 500 μm. (C) The results of tube formations of HUVECs after different treatments. Scale bars: 100 μm. (D) Analysis of the wound-healing area rates at 10 h. Data are presented as mean ± SD (n = 3). (E) Analysis of the tube lengths. Data are presented as mean ± SD (n = 3). One-way ANOVA method was applied for statistical analysis ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant.

Figure 3

AAV2-SPLTH inhibits fluorescence leakage (A) Representative pictures of FFA in different groups after different treatments. (B) The quantitative statistical plot of (A) by scoring the fluorescence leakage in each eye. Data are presented as mean ± SD (n = 6). (C) Representative images of H&E staining after different treatments. (D) Statistics of the number of cells in the ONL layer in (B). Data are presented as mean ± SD (n = 6). One-way ANOVA method was applied for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant.

Figure 4

AAV2-SPLTH could not completely save photoreceptor cell layer death (A) TUNEL assay determined the effect of AAV2-SPLTH on cell apoptosis after 10 days of doxycycline (Dox) induction. (B) TUNEL assay determined the effect of AAV2-SPLTH on cell apoptosis after 30 days of Dox induction. (C) Analysis of the TUNEL-positive cell rates. Data are presented as mean ± SD (n = 6). One-way ANOVA method was applied for statistical analysis, and data are mean ± standard error. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant.

Figure 5

Proteomic analysis of AAV2-SPLTH–mediated antiangiogenic effects in Tet-opsin-VEGFA mice (A) Volcano plot shows proteins that differ significantly between untread and Dox groups based on fold change and p value. In particular, blue (fold change < 0.67; p < 0.05) or red (fold change >1.5; p < 0.05) dots indicate the presence of significantly downregulated or upregulated metabolites, respectively. Gray dots were non-significantly different compounds. n = 3. (B) Volcano plot shows protein that differ significantly between Dox and AAV2-SPLTH groups. (C) UpSet Venn diagram showed differential protein intersection analysis in untreated group, Dox group, and AAV2-SPLTH group. The green box is the saved protein set of AAV2-SPLTH, and the orange box is the unsaved protein set of AAV2-SPLTH. (D) KEGG enrichment analysis of AAV2-SPLTH salvage protein collection. (E) KEGG enrichment analysis of AAV2-SPLTH unsalvaged protein collection.

Figure 6

Immunofluorescence staining in the eyes of Tet-opsin-VEGFA mice (A) Immunofluorescence staining of RCVRN on retina sections. (B) Immunofluorescence staining of RCVRN on retina sections. Scale bars: 50 μm.

Figure 7

Evaluation of protein expression and ERG in the eyes of Tet/opsin/VEGFA mice (A) Western blot analysis of the Rho expression levels (actin was used as an internal control). (B) The relative protein expression intensity of Rho. Data are presented as mean ± SD (n = 3). (C) Western blot analysis of the RCVRN expression levels (actin was used as an internal control). (D) The relative protein expression intensity of RCVRN. Data are presented as mean ± SD (n = 3). (E) Scotopic ERG showing A-wave or B-wave amplitudes at 10 days after Dox induction (n = 6). (F) Scotopic ERG showing A-wave or B-wave amplitudes at 30 days after Dox induction (n = 6). One-way ANOVA method was applied for statistical analysis, and data are mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant.