Abstract
Purpose :
Current therapies that target vascular complications of DR are only effective in 25-50% of DR patients. New strategies, including targeting the CCL2-CCR2 pathway, are being explored. CCL2 is a chemokine that mediates myeloid cell recruitment from the circulation into the central nervous system, but its function in the retina has not been well characterized. In this study we investigate the role of CCL2 in ocular neovascularization (NV) using a murine model of ischemic retinopathy.
Methods :
Retinas from C57BL/6 mice with oxygen-induced retinopathy (OIR) were collected at several time points and Ccl2 expression levels were measured using qPCR (age-matched normoxia mice were used as controls). To test the role of CCL2 in OIR pathogenesis, varying doses of recombinant murine CCL2 (rmCCL2; 0-632nM) were injected intravitreally at p12. Neovascularization (NV) was assessed by immunohistochemistry (IHC) at p17. Flow cytometry was used to assess myeloid cell recruitment and to assess phagocytic function of microglia/macrophages by measuring uptake of fluorescent beads by CD11b+ cells, after rmCCL2 or PBS injection. The effects of CCL2 neutralizing antibodies were studied by IHC.
Results :
OIR mice had significantly higher levels of retinal Ccl2 at p17 and p21 than normoxia controls. Intravitreal injection of rmCCL2 at p12 significantly prevented NV in OIR in a dose-dependent manner. Interestingly, 15.5nM of CCL2 provided the strongest effect; while lower and higher doses were significantly less effective. The degree of CCL2-induced rescue of OIR was not proportional to the numbers of retinal CD11b+ cells, but rather,the degree of phagocytic function. Finally, CCL2 neutralization at p17 induced severe hemorrhaging and NV at p19.
Conclusions :
The role of the CCL2-CCR2 signaling pathway in the retina is probably more complicated than we previously thought. Our results show that CCL2 potently prevents NV in OIR due to changes in microglia/macrophage function rather than enhanced myeloid cell recruitment. In contrast, neutralizing CCL2 induces severe NV. This work supports a paradigm shift for the manner in which CCL2 is targeted in DR patients, since CCL2 supplementation might provide enhanced therapeutic benefits.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.