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E. Sakurai, M. Nozaki, B.J. Raisler, J.Z. Baffi, Y. Ogura, B.K. Ambati, J. Ambati; Inhibition of Choroidal Neovascularization by VEGF–A Blockade Is Mediated via Ccl–2 Suppression . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3027.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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