Anti-Syndecan 2 Antibody Treatment Reduces Edema Formation and Inflammation of Murine Laser-Induced CNV

Abstract

Purpose: Alteration of visual acuity in wet age-related macular degeneration (AMD) is mostly driven by vascular endothelial growth factor A (VEGF-A)-induced edema from leaky newly forming blood vessels below the retina layers. To date, all therapies aimed at alleviation of this process have relied on inhibition of VEGF-A activity. Although effective in preventing vascular leak and edema, this approach also leads to the loss of normal vasculature and multiple related side effects.

Methods: We have developed an alternative strategy that uses anti-syndecan-2 polyclonal antibody (anti-Sdc2 pAb) to block VEGF-A-induced permeability without interfering with other VEGF-A activities. The effect of anti-Sdc2 pAb therapy was assessed in vitro using a transendothelial electrical resistance (TEER) assay, as well as staining of the endothelial cell junction, and in vivo in the laser-induced choroidal neovascularization (CNV) model.

Results: Anti-Sdc2 pAb blocked VEGF-A-induced permeability in vitro, and both local intravitreal injections and systemic intravenous treatments with anti-Sdc2 pAb were as effective as intravitreal anti-VEGF therapy in reducing edema, size of retinal lesions, and local inflammation in this model. Post-injury neovascularization was not affected by treatment with anti-Sdc2 pAb.

Conclusions: These findings indicate that anti-Sdc2 pAb therapy can be an effective alternative to anti-VEGF-A approaches for suppression of edema and to prevent retinal lesions in wet neovascular AMD (nAMD).

Translational relevance: Intravitreal anti-Sdc2 treatment may avoid side effects observed with the long-term anti-VEGF therapy, and systemic treatment with an anti-Sdc2 pAb antibody can address the issues associated with repeated intravitreal injections.

Figure 1.

Electroretinographic recording shows no abnormalities after anti-Sdc2 pAb administration in healthy mice that were not treated with laser. Either vehicle (PBS) or anti-Sdc2 pAb was administered to healthy mice via intravitreal injection, and ERG recording was performed on day 8. (A–C) Mixed cone and rod responses recorded at light intensities of −0.5, 0, and 1 log cd·s/m² (a representative sweep for each group is shown). (D–I) Quantification of a- and b-wave amplitudes at different flash strengths indicated no differences in a- or b-waves between PBS- and Sdc2 pAb–treated eyes. Graphs are presented as scatterplots with mean ± SEM (n = 10 vehicle, n = 9 Sdc2 pAb–treated eyes). Data were analyzed by unpaired two-tailed Student's t-test.

Figure 2.

Anti-Sdc2 pAb administration effectively reduces CNV leakage and lesion size. FA was performed on day 7 after laser photocoagulation. (A–E) Illustrative FA images of eyes intravitreally injected with vehicle, anti-Sdc2 pAb, or anti-VEGF or systemically treated (vehicle, anti-Sdc2 pAb). (F) Quantification of leakage area by FA of eyes intravitreally injected with vehicle (n = 34 lesions), anti-Sdc2 pAb (n = 30 lesions), or anti–VEGF-A (n = 18 lesions) or systemically treated with vehicle (n = 23 lesions) or anti-Sdc2 pAb (n = 20 lesions). (G–K) Illustrative en face OCT images of eyes intravitreally injected with vehicle, anti-Sdc2 pAb, or anti-VEGF or systemically treated with vehicle or Sdc2 pAb. (L) Quantification of lesion volume by SD-OCT of eyes intravitreally injected with vehicle (n = 33 lesions), anti-Sdc2 pAb (n = 29 lesions), or anti-VEGF (n = 41 lesions) or systemically treated by vehicle (n = 30 lesions) or anti-Sdc2 pAb (n = 31 lesions). Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Analysis of RPE complexes. (A–E) Immunocytochemistry analysis of mice retinas 8 days after indicated treatments. (F) CD31 staining and associated quantification of CD31 staining (n = 12 lesions). (G–L) ERG staining (endothelial marker) (G–K) and associated quantification (L) (n = 12–16 lesions). (M–R) F4/80 staining (macrophage marker) (M–Q) and associated quantification (R) (n = 12 lesions). Data are presented as column (mean) and mean ± SEM and analyzed by one-way ANOVA followed Šidák multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Sdc2 pAb restores endothelial barrier function in mRECs treated with VEGF-A. (A) Confluent mRECs were stimulated for 12 hours with VEGF-A or vehicle, which led to disruption of the endothelial barrier, as measured by a drop in normalized cell index (green vs. magenta line). Administration of anti-Sdc2 pAb at 12 hours after VEGF-A led to concentration-dependent recovery of endothelial barrier integrity (red, cyan, and blue lines) compared to treatment with vehicle alone (green line). (B) Quantification of the differences in cell index 24 hours after initiation of anti-Sdc2 pAb treatment. (C–H) Staining with junction markers (VE-cadherin and ZO-1) of mRECs stimulated with VEGF-A for 12 hours and then treated with either vehicle or Sdc2 pAb for 24 hours. (I, J) Assessment of VEGFA-induced proliferation in the presence of Sdc2 pAb versus vehicle. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA with Šidák multiple-comparison test (A, B, I, J).