Chemokines and Their Corollary Receptors Regulate Retinal Angiogenesis in a Mouse Model of ROP

Abstract

Purpose: : A growing body of evidence implicating inflammation in vasoproliferative ocular disease has spurred interest in the role of immunity in the genesis and persistence of these diseases. The C-C family of chemokines induces chemotaxis in several types of leukocytes (a cardinal feature of retinopathy) to sites of injury in vivo. To this end, we asked what role the C-C chemokine family plays in pathological angiogenesis during oxygen-induced retinopathy (OIR).

Methods: : C57Bl/6, Ccl2-/-, Ccl3-/-, Ccr2-/-, and Ccr5-/- mice were subjected to OIR (75% oxygen from P7-P12) and eyes were harvested at P17, P21, and P25. Disease severity was quantified at each time-point. Lasercapture microdissection (LCM) was used to isolate normal retinal vessels and pathological neovascularization (NV) and chemokine expression profiles were compared using quantitative real-time PCR. Chemokines and microglia were localized with immunohistochemistry (IHC) and the number of NV associated microglia was quantified in each strain. Additionally, the chemotactic effect of Ccl2 and Ccl3 on isolated retinal microglia was analyzed in vitro.

Results: : 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 increased NV at P17H compared to wild-type controls, while Ccl2-/- mice show normal NV at P17, but delayed NV regression (7.9 ± 1.0% and 3.6 ± 0.9% vs. 3.6 ± 0.5%; P < 0.001 and 0.8 ± 0.2%; P < 0.01). LCM RNA profiles show significant upregulation of both Ccl2 and Ccl3 in pathological NV vs. normal vessels. Ccl2 and Ccl3 co-localize with NV using IHC, and the C-C family receptors, Ccr2 and Ccr5, localized to neovessel associated macrophages. Consistent with these data, Ccl2 and Ccl3 induce the chemotaxis of retinal microglia in vitro, and Ccl2 and Ccl3 null mice have significantly fewer NV associated microglia in vivo at P17 (11.9 ± 0.8, 9.17 ± 0.9, vs 21.5 ± 1.4, P < 0.0001).

Conclusions: : The chemokines Ccl2, Ccl3 and the receptors Ccr2 and Ccr5 help modulate the angiogenic response during OIR. Our data suggests secretion of Ccl2 and Ccl3 by pathological neovessels and consequent recruitment of chemokine receptor positive macrophages. Increased retinal NV and slower NV regression in chemokine/chemokine receptor null animals suggests a role for these cytokines in limiting pathologic NV during OIR.

Keywords: retinal neovascularization • inflammation • retinopathy of prematurity