Differential Influences of CCL2/CX3CR1 Axis on Vascular and Retinal Degeneration in Diabetic Retinopathy

Abstract

Purpose : CCL2-CCR2 and CX3CL1-CX3CR1 are central chemokine axes that regulate the recruitment of monocytes/macrophages during acute or chronic tissue injury. Enhanced macrophage infiltration and microglial activation in the retina have been linked to vascular leakage in early diabetic retinopathy. Using Ccl2/Cx3cr1 double knockout (DKO) mice on C57BL/6J background (without rd8 mutation), we investigate the role of CCL2-CCR2 and CX3CL1-CX3CR1 pathways in retinal microglia/macrophage activation and neurovascular degeneration in diabetic retinopathy.

Methods : Diabetes was induced by streptozotocin in adult wildtype (WT) or DKO mice and maintained for 5 months after the onset of diabetes. Retinal neuronal and vascular changes were evaluated by color fundus imaging, fluorescence angiography, TUNEL assay, histological and morphometric analysis. Retinal inflammation and microglial activation were examined by immunofluorescence staining, western blotting, and macrophage-endothelial adherence assay using brain microvascular endothelial cells and bone marrow derived macrophages isolated from WT or DKO mice.

Results : Diabetic DKO mice exhibited markedly increased number of focal retinal degenerative lesions accompanied by augmented apoptosis of photoreceptor cells compared to diabetic WT mice. Fluorescence angiography demonstrated tortuous and beading retinal blood vessels in diabetic WT mice, but to a much less extent in diabetic DKO mice, suggesting that these mice are protected from diabetes-induced vascular damage. Further, diabetic DKO mice showed enhanced activation and altered distribution of microglia toward the outer retina, increased retinal VEGF and TNF-α expression but decreased NF-κB activation and reduced VCAM-1 expression. In vitro study confirmed that macrophages derived from DKO mice had significantly reduced adherence ability to WT or DKO endothelial cells. In contrast, there was only a modest decrease in WT macrophage adherence to DKO endothelial cells compared to the WT control.

Conclusions : We conclude that CCL2/CX3CR1 deficiency exacerbates outer neural retinal degeneration but attenuates vascular damage in diabetic retinopathy. These results may also suggest a differential role of macrophages and microglia in mediating vascular and neuronal injury in retinal diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.