Chemokine Ligands And Receptors Mediate Pathological Retinal Neovascularization In Oxygen-induced Retinopathy

Abstract

Purpose: : Pathologic retinal neovascularization is the primary cause of blindness in proliferative retinopathy. Recent evidences implicate increasing interest in the roles of inflammation and immunity in the pathogenesis of retinopathy. The C-C subgroup of chemokines induces chemotaxis of inflammatory cells to retinal cites of injury (a cardinal feature of retinopathy). In this study we assessed the role of chemokines (Ccl2 and Ccl3) and their receptors (Ccr2 and Ccr5) in pathological retinal angiogenesis during oxygen-induced retinopathy (OIR) using transgenic mice lacking these molecules.

Methods: : Ccl2^-/-, Ccl3^-/-, Ccr2^-/-, Ccr5^-/- and wild type (WT) control mice were subjected to OIR (75% oxygen from postnatal day (P) 7-P12). Eyes were dissected at P17, P21 and P25, and disease severity was quantified. Normal retinal vessels and pathological neovascularization (NV) were isolated with laser capture microdissection (LCM), and chemokine expression levels were compared using RT-qPCR. Boyden chambers were used to analyze the chemotactic effect of Ccl2 and Ccl3 on isolated retinal microglia in vitro.

Results: : At P17 after OIR, Ccl3^-/- (11.6 ± 0.4% vs. 8.3 ± 0.6%; P ≤ 0.0001), Ccr2^-/- (14.6 ± 0.6 vs. 7.8 ± 0.4%, P ≤ 0.0001), and Ccr5^-/- (14.0 ± 0.7 vs. 7.8 ± 0.4%; P ≤ 0.0001) mice have significantly increased NV as compared to WT controls. In contrast, Ccl2^-/- mice show increased NV at P21 (7.9% ± 1.0 vs. 3.6% ± 0.5; P ≤ 0.001) and P25 (3.6% ± 0.9 vs. 0.8% ± 0.2; P ≤ 0.01) after exhibiting normal NV at P17. Both Ccl2 and Ccl3 are upregulated in and colocalize with pathological NV as compared to normal vessels. Their receptors, Ccr2 and Ccr5, are localized to neovessel associated macrophages. In addition, Ccl2 and Ccl3 induce the chemotaxis of retinal microglia in vitro, and the numbers of NV associated microglia in vivo are significantly decreased in Ccl2 and Ccl3 null mice at P17.

Conclusions: : Ccl3 and its cognate receptor attenuate NV during OIR while Ccl2 is necessary for the timely resolution of NV in OIR. Our data suggests neovessels likely secrete both Ccl2 and Ccl3 and consequently recruit chemokine receptor positive macrophages to modulate retinal angiogenic response, indicating a role for these cytokines in limiting pathologic NV during OIR.

Keywords: retinal neovascularization • cytokines/chemokines