The role of M2 macrophages in promoting ischemia-induced retinal neovascularization in mice

Abstract

Purpose: M2 macrophages are regarded to play important roles in angiogenesis, and we previously demonstrated that the M2 macrophage marker overexpressed in vitreous and fibrovascular membranes (FVM) of proliferative diabetic retinopathy (PDR) patients, but its pathogenesis in retinal neovascularization still remains unclear. The purpose of this study was to investigate the role of M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR).

Methods: OIR was induced in C57BL/6J mice by exposing postnatal day (P)7 pups to 75% oxygen for 5 days and then returning them to room air at P12. Eyes were enucleated at P12 to P25. CD206 was used as an M2 macrophage marker, and real-time RT-PCR and immunofluorescence staining were used to access the levels and distributions of M2 macrophages. M2 macrophages were selectively depleted by mannosylated clodronate liposomes (MCLs). MCLs, bone marrow-derived M2 macrophages and their controls were injected into the vitreous at P12 to examine the effects at P17. Avascular areas and neovascular tufts areas were evaluated by immunohistochemical analysis. Bone marrow-derived M2 macrophages were co-cultured with human retinal endothelial cells (HRECs) to examine the effects on proliferation and tube formation.

Results: The results show that the mRNA expression of M2 macrophage marker as well as the number of M2 macrophages increased significantly in OIR retinas compare to the room air controls (P<0.05). Distributions of M2 macrophages were closely associated with neovascular tufts in OIR mice. Selective depletion of M2 macrophages suppressed the pathological neovascularization (35.8% reduction, P<0.01) and promoted physiological revascularization (18.8% reduction, P<0.05). In contrast, intravitreal injection of bone marrow-derived M2 macrophages promoted pathological neovascularization (53.5% increase, P<0.01) and inhibited physiological revascularization (21.2% increase, P<0.05). Both VEGF-A (P<0.01) and Hif-1α (P<0.05) were reduced by selective depletion of M2 macrophages. In a vitro co-culture system, M2 polarized macrophages significantly promoted proliferation and tube formation of HRECs (P<0.01).

Conclusions: These results indicate that M2 macrophages play an important role in promoting ischemia-induced neovascularization in the retina. Targeting M2 macrophages thus could be a potential therapeutic option for inhibiting retinal pathological neovascularization.