Recombinant human VEGF165b inhibits experimental choroidal neovascularization

Abstract

Purpose: Vascular endothelial growth factor (VEGF-A) is the principal stimulator of angiogenesis in wet age-related macular degeneration (AMD). However, VEGF-A is generated by alternate splicing into two families, the proangiogenic VEGF-A(xxx) family and the antiangiogenic VEGF-A(xxx)b family. It is the proangiogenic family that is responsible for the blood vessel growth seen in AMD.

Methods: To determine the role of antiangiogenic isoforms of VEGF-A as inhibitors of choroidal neovascularization, the authors used a model of laser-induced choroidal neovascularization in the mouse eye and investigated VEGF-A(165)b effects on endothelial cells and VEGFRs in vitro.

Results: VEGF-A(165)b inhibited VEGF-A(165)-mediated endothelial cell migration with a dose effect similar to that of ranibizumab and bevacizumab and 200-fold more potent than that of pegaptanib. VEGF-A(165)b bound both VEGFR1 and VEGFR2 with affinity similar to that of VEGF-A(165). After laser injury, mice were injected either intraocularly or subcutaneously with recombinant human VEGF-A(165)b. Intraocular injection of rhVEGF-A(165)b gave a pronounced dose-dependent inhibition of fluorescein leakage, with an IC(50) of 16 pg/eye, neovascularization (IC(50), 0.8 pg/eye), and lesion as assessed by histologic staining (IC(50), 8 pg/eye). Subcutaneous administration of 100 microg twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size.

Conclusions: These results show that VEGF-A(165)b is a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition.

Figure 1.

VEGF-A₁₆₅b is the predominant VEGF-A_xxxb isoform in the eye. Protein was extracted from dissected tissues of donor eyes from the Bristol Eye Bank and subjected to SDS-PAGE and immunoblotting using anti-VEGF (left) or anti-VEGF_xxxb (right). Protein was extracted from eyes either by dissection of the RPE layer from the rest of the retina or by dissection of the neuronal layer from the rest of the retina or the whole retina. Recombinant human (rh) VEGF₁₆₅ and VEGF₁₆₅b were run both as denatured and native to demonstrate monomer and dimers. The expected sizes of VEGF₁₂₁, VEGF₁₈₉, and VEGF₁₄₅ are demonstrated.

Figure 2.

VEGF-A₁₆₅b inhibits VEGF-A₁₆₅–mediated endothelial cell migration but binds to VEGFR. (A) HMVECs were seeded onto polycarbonate 8-μm pore filters, and migration across the transwell was measured to 1 nM VEGF-A₁₆₅ with increasing doses of inhibitors. (B) Fc-VEGFR1 was coated onto an ELISA plate, and increasing concentrations of biotinylated VEGF-A₁₆₅b were added. After development with HRP-SA, the percentage of bound VEGF-A₁₆₅b was calculated relative to saturated values, and IC₅₀ was calculated using a nonlinear sigmoid variable slope fit. (C) Fc-VEGFR1 was coupled to an SPR sensor chip, and increasing concentrations of VEGF-A₁₆₅b were allowed to flow over the chip. Response units relative to the reference chip were measured by surface plasmon resonance. (D) Fc-VEGFR2 was coupled to a similar chip, and the experiment was repeated. Affinity and dissociation constants were calculated by 1:1 Langmuir binding models.

Figure 3.

Intravitreal VEGF-A₁₆₅b inhibits CNV. Mice underwent retinal laser coagulation in both eyes and intravitreous injection with VEGF-A₁₆₅b or HBSS directly after laser procedure (day 0) and on day 7. (A) FA on day 14 showed dose-dependent inhibition of leakage (arrows). (B) Quantification using 0 to 3 scoring scheme by three independent masked observers. (C) Staining with isolectin B4 of the RPE-choroid-sclera complex showed a dose-dependent decrease in lesion size. (D) The IC₅₀ for lesion size was 0.78 pg. (E) H&E staining of the middle section of each photocoagulation area showed disrupted RPE layer and lesions. These were dose dependently reduced by VEGF-A₁₆₅b injection. (F) Measurement of the lesion size.

Figure 4.

Systemic VEGF-A₁₆₅b inhibits CNV. Mice underwent retinal laser coagulation in both eyes and subcutaneous injection with 100 μg VEGF-A₁₆₅b or PBS twice weekly. (A) Quantification of FA on day 14 using 0 to 3 scoring scheme by three independent masked observers showed inhibition of leakage. Representative images for each treatment are shown above the bars. (B) Staining with isolectin B4 of the RPE-choroid-sclera complex. (C) Confocal three-dimensional reconstruction was used to calculate volume size. (D) H&E staining of the middle section of each photocoagulation area showed disrupted RPE layer and lesions. These were reduced by VEGF-A₁₆₅b injection. **P < 0.02 and ***P < 0.001 compared with PBS. Unpaired t-test (A–C). Mann-Whitney U test (D).