Excess Endogenous VEGF–A Suppresses Laser–induced Choroidal Neovascularization via VEGFR–1

Abstract

Abstract: : Purpose: To determine the mechanistic basis underlying the clinical observation that despite widespread disease in patients with age–related macular degeneration (AMD) there is often but a single focus of CNV, and that CNV often recurs soon after ablation, and to test the hypothesis that endogenous vascular endothelial growth factor–A (VEGF–A) establishes this zone of inhibition in the laser–injury model. Methods: CNV was induced by laser injury in C57BL/6J and Vegfr–1 tyrosine kinase^–/– mice, and volumes measured 7 days later by confocal evaluation of Griffonia simplicifolia Isolectin B4 staining of RPE–choroid flamounts. CoCl₂, a chemical mimetic of hypoxia, was injected into the vitreous following injury to upregulate VEGF–A. Endogenous VEGF–A also was increased by placing laser spots 2 days or 2 weeks prior to the placement of subsequent laser spots whose CNV response was evaluated. Neutralizing VEGF–A, VEGFR–1, VEGFR–2, or control antibodies were injected into the vitreous following injury. Results: Intravitreous injection of CoCl₂ increased VEGF–A levels in the RPE/choroid, which peak 3 days after laser injury, by 88±27% compared to PBS (P < 0.01). CoCl₂ decreased laser–induced CNV by 61±10% (P < 0.01). Neutralizing antibodies to VEGF–A and VEGFR–1 restored CNV that was inhibited by CoCl₂ (89±13% and 126±23% of control respectively); however, VEGFR–2 blockade did not abrogate CoCl₂–induced CNV suppression (46±10% of control). Preexisting injury suppressed CNV volume by 50±8% (P < 0.01) if placed 2 days earlier but not when placed 2 weeks earlier. This zone of inhibition was pronounced in injured areas closer to (600 µm) the preexisting injury than in areas further from (1000 µm) it. Neutralizing VEGF–A antibody but not control IgG abolished the suppression of CNV by preexisting injury (P < 0.05). CNV volume was increased by 37±13% (P < 0.05) in Vegfr–1 tyrosine kinase^–/– mice compared to wild–types. Conclusions: Collectively these findings demonstrate that excess endogenous VEGF–A can suppress laser–induced CNV via VEGFR–1 signaling, and support the contention that VEGF–A establishes a zone of angiogenic inhibition. These data also may be used to better guide timing and dosage strategies of anti–VEGF–A therapies in AMD.

Keywords: choroid: neovascularization • growth factors/growth factor receptors • age-related macular degeneration