Figure 2.
Macrophages are required for the early wound healing response in CNV lesions. A and B: Cell-fate tracing experiments in laser-induced CNV lesions. EYFP expression in cells of the myeloid lineage (lysozyme M Cre R26-stopfl/fl-EYFP floxed mice) and colabeling for the vascular endothelial cell marker CD144 (VE-cadherin) showed that myeloid cells do not transdifferentiate into vascular endothelial cells. No colabeling was observed between EYFP and CD144. A shows a three-dimensional reconstruction of confocal microscopy images of a CNV lesion from these mice. C: Immunolabeling for F4/80 (macrophages) and CD144 (endothelial cells) showed that macrophages and endothelial cells are both present in close proximity within CNV lesions, but no cells coexpressed both cell markers. D: Cell-fate tracing experiments in mice that express EYFP in RPE cells (arrow) showed that the SMA+ fibroblastic-scaffold (arrowhead) is not derived from RPE cells (EYFP−). E: At 66 hours after laser injury, no CD31+ endothelial cells have infiltrated the site of laser injury, whereas the SMA+ myofibroblastic cells exhibit strong staining for NG2 at the site of laser injury. F: In fully formed CNV lesions at day 5, a prominent SMA+NG2+ myofibroblastic scaffold persists, whereas F4/80+ macrophages fully populate the site of laser injury. G: SMA+NG2+ cells participate in the myofibroblastic scaffold and also surround formed neovessels, consistent with a pericyte-like function. H and I: While RPE cells adjacent to the laser injury site were SMA+NG2−, the myofibroblastic scaffold was demarcated by SMA+NG2+ staining. J: Macrophages remain an abundant cell population even after the formation of neovessels. Three-dimensional reconstruction of confocal images. K: The area of neovascularization does not extend beyond the area of the SMA+ myofibroblastic scaffold. L–N: Ablation of macrophages in Itgam-Dtr mice with injection of diphtheria toxin at days −1 and +1 after laser injury showed that effective attenuation of macrophages correlates with inhibition of the formation of the fibrovascular scaffold and SMA expression in adjacent RPE cells. Original magnification: ×10 (F–I, K); ×20 (A, B, E, J, L–N); ×40 (C, D).