Inhibition of Choroidal Neovascularization by VEGF–A Blockade Is Mediated via Ccl–2 Suppression

Abstract

Abstract: : Purpose: To determine whether inhibition of choroidal neovascularization (CNV) by vascular endothelial growth factor–A (VEGF–A) blockade is mediated via suppression of monocyte chemoattractant protein–1 (Ccl–2/MCP–1). Methods: CNV was induced by laser injury in C57BL/6J and Ccl2^–/– mice and volumes measured 7 days later by confocal evaluation of Griffonia simplicifolia Isolectin B4 staining of RPE–choroid flamounts. Neutralizing VEGF–A, Ccl–2, or control antibodies, and recombinant VEGF–A₁₆₄ or Ccl–2 were injected into the vitreous following laser injury. Ccl–2 and VEGF–A levels in the RPE/choroid were measured by ELISA and macrophage infiltration into the choroid was analyzed by flow cytometry gating for F4/80⁺CD11c^– cells. Results: Peak Ccl–2 levels following laser injury exceeded peak VEGF–A levels by 247±47% (P < 0.01). Laser injury recruited macrophage infiltration into the choroid, peaking 3 days after injury. Injected on days 0 and 1, neutralizing VEGF–A antibody significantly reduced CNV (55±13%; P = 0.04) compared to PBS. However, when injected on days 2 and 3, the inhibition was modest and insignificant (33±8%; P = 0.07). When injected on days 4 and 5, the inhibitory effect was lost (7±19%; P = 0.79). CNV was inhibited by VEGF–A neutralization only before macrophage recruitment, and closely paralleled decreased macrophage recruitment (r² = 0.8; P = 0.03). VEGF–A antibody, injected on days 0 and 1, decreased Ccl–2 protein in the RPE/choroid by 41±11% (P = 0.05) and macrophage infiltration Recombinant Ccl–2 restored the CNV inhibited by VEGF–A antibody. CNV and macrophage infiltration was reduced in wild–type mice treated with Ccl–2 antibody and in Ccl2^–/–mice by greater than 75%. Recombinant VEGF–A₁₆₄ could not restore CNV in Ccl2^–/– mice or wild–type mice treated with Ccl–2 antibody. Conclusions: Collectively these data demonstrate that VEGF–A supports CNV indirectly via stimulation of Ccl–2. Given the growing recognition of VEGF–A as a neuroprotective factor, our data suggest that Ccl–2 blockade may achieve similar or greater anti–angiogenic effect without compromising neural function dependent on VEGF–A.

Keywords: age-related macular degeneration • choroid: neovascularization • cytokines/chemokines