Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar

Abstract

After wounding, multiple cell types interact to form a fibrovascular scar; the formation and cellular origins of these scars are incompletely understood. We used a laser-injury wound model of choroidal neovascularization in the eye to determine the spatiotemporal cellular events that lead to formation of a fibrovascular scar. After laser injury, F4/80(+) myeloid cells infiltrate the wound site and induce smooth muscle actin (SMA) expression in adjacent retinal pigment epithelial cells, with subsequent formation of a SMA(+)NG2(+) myofibroblastic scaffold, into which endothelial cells then infiltrate to form a fibrovascular lesion. Cells of the fibrovascular scaffold express the proangiogenic factor IL-1β strongly, whereas retinal pigment epithelial cells are the main source of VEGF-A. Subsequent choroidal neovascularization is limited to the area demarcated by this myofibroblastic scaffold and occurs independently of epithelial- or myeloid-derived VEGF-A. The SMA(+)NG2(+) myofibroblastic cells, F4/80(+) macrophages, and adjacent epithelial cells actively proliferate in the early phase of the wound healing response. Cell-lineage tracing experiments suggest that the SMA(+)NG2(+) myofibroblastic scaffold originates from choroidal pericyte-like cells. Targeted ablation of macrophages inhibits the formation of this fibrovascular scaffold, and expression analysis reveals that these macrophages are Arg1(+)YM1(+)F4/80(+) alternatively activated M2-like macrophages, which do not require IL-4/STAT6 or IL-10 signaling for their activation. Thus, macrophages are essential for the early wound healing response and the formation of a fibrovascular scar.

Figure 1

Formation of a fibrovascular network after laser injury. A: At the site of laser injury (asterisk), F4/80⁺ macrophages (arrowheads), are seen within 24 to 40 hours. B: Adjacent to infiltrating F4/80⁺ macrophages (arrowhead), RPE cells exhibit increased SMA expression (arrow) at approximately 48 hours after laser injury (asterisk). C: SMA⁺ stellate-like cells (arrow) are seen originating from the center of laser spots at approximately 60 hours after injury (asterisk). At that time RPE cells (arrowhead) adjacent to the injury site exhibit strong SMA expression. D: At approximately 72 hours after laser injury, F4/80⁺ macrophages (arrowhead) are the predominant cell population within the area of injury (asterisk); SMA⁺ cells are also present (arrow). E: At approximately 72 hours, a SMA⁺ myofibroblastic scaffold is seen and adjacent RPE cells are SMA⁺, but endothelial cells have not yet infiltrated the site of laser injury. F: At approximately 96 hours, CD31⁺ neovessels (arrow) can be observed at the center of the site of laser injury. G and H: Between day 4 and 5 after laser injury, a fully formed fibrovascular lesion can be observed, with an extensive network of CD31⁺ neovessels (arrow). I and J: Confocal microscopy of a fully formed CNV lesion at day 10 after laser injury shows that neovessels are covered and demarcated by a SMA⁺ myofibroblastic network. Neovascularization does not extend beyond the SMA⁺ myofibroblastic network. K and L: Top (K) and midportion (L) of lesion at 10 days after laser injury. M: SMA expression is maintained in RPE cells adjacent to the site of laser injury, even 4 months after injury. N–Q: Staining of a CNV lesion at 72 hours after laser injury for the proliferation marker phospho-histone H3 (Ser10) exhibits extensive proliferation of multiple cell types within an early CNV lesion (N). Some F4/80⁺ cells stain strongly for this proliferation marker (O), as well as stellate SMA⁺ myofibroblastic cells (P) and adjacent SMA⁺ RPE cells (Q). Original magnification: ×10 (E–I, M, and N); ×20 (A–D); ×40 (K, L, O–Q).

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-stop^fl/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).

Figure 3

Macrophages in laser-induced CNV lesions are alternatively activated, independent of IL-4/STAT6 or IL-10 signaling. A: Western blotting of choroidal tissue lysates reveals up-regulation of the M2-type marker Arg1 at day 3 after laser-induced wound injury. B: Immunolabeling for F4/80 and Arg1 shows that in CNV lesions the majority of macrophages are Arg1⁺ (arrow) at approximately 72 hours after injury; however, quiescent choroidal macrophages do not express Arg1 (arrowhead). C: Macrophages infiltrating CNV lesions exhibit colabeling for F4/80 and Arg1 (whole-mount confocal microscopy image). D: In control mice, macrophages in fully formed CNV lesions are F4/80⁺Arg1⁺. E and F: In Stat6^−/− macrophages (F4/80⁺) can be seen in laser-induced CNV lesions to express YM1 (E) and Arg1 (F). G: In IL-10 null mice (Il10^−/−) Arg1⁺ F4/80⁺ macrophages are present in laser-induced CNV lesions. H: Arg1 protein levels are increased at day 3 after wound injury in choroidal lysates wild-type mice, as well as in Stat6^−/− and Il10^−/− mice. Depletion of myeloid cells (DTR⁺) prevents the increase of choroidal Arg1 levels, demonstrating that the observed arginase increase is due to macrophage infiltration. DTR−, Dtr^fl/fl mice treated with diphtheria toxin; DTR+, lysozyme M Cre Dtr^fl/fl mice treated with diphtheria toxin. Original magnification: ×10 (B); ×20 (C–G).

Figure 4

Infiltrating wound macrophages express markers of M2-type macrophages. Semiquantitative RT-PCR of RPE–choroid tissue lysates obtained immediately after laser injury or 3 days later. A and B: The pan-macrophage markers F4/80 (A) and CD68 (B) were significantly increased 3 days after laser injury in wild-type mice and in IL-10 and STAT6 null mice. Relative mRNA levels are indicated normalized to m36B4 levels. C–F: The M1-type macrophage marker iNOS (C) was down-regulated 3 days after laser injury (normalized to either m36B4 or to the total macrophage population using F4/80 mRNA levels), whereas the M2-type markers Arg1 (D), CCL17 (E), and IL1RA (F) were significantly up-regulated. Data are expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

Expression of VEGF-A and IL-1β in CNV lesions. A: Removal of RPE cells in choroidal flat mounts (top part of A), shows that only RPE cells express VEGF-A (β-gal⁺), while underlying choroidal cells are β-gal⁻. A and B: In adult RPE cells (arrow), VEGF-A is strongly expressed, although the underlying choroidal cells exhibit no evidence of VEGF-A expression in Vegfa^lacZKI/WT mice. C: Expression of the VEGF-A receptor Flt1 is prominent in quiescent choroidal vessels, shown here by β-gal staining in Flt1^lacZKI/WT mice. D: Similarly, expression of the VEGF-A receptor Flk1 can be seen in choroidal vessels, shown here by β-gal staining in Flk1^lacZKI/WT mice. E–H: In immunolabeling experiments for β-gal in Vegfa^lacZKI/WT mice, nuclear β-gal staining identifies the cells that express VEGF. E: Blood vessels and the SMA⁺ myofibroblastic cells within CNV lesions exhibit no strong VEGF-A expression (β-gal⁻), whereas the adjacent RPE cells exhibit strong VEGF-A expression (β-gal⁺). F: No VEGF-A signal was seen in F4/80⁺ macrophages in CNV lesions. G: Although F4/80⁺ macrophages (arrow) exhibit no nuclear β-gal staining, adjacent RPE cells exhibit strong VEGF-A expression (arrowhead). H: No VEGF-A expression is seen within CNV lesions in Vegfa^lacZKI/WT mice. At 72 hours after laser injury, infiltrating macrophages exhibit no IL-1β expression or expression only in a few cells, whereas the non-myeloid cells in the CNV lesions exhibit strong expression for IL-1β. I and J: Conditional inactivation of VEGF-A in RPE cells postnatally shows that fully formed neovascular lesions can form despite Cre expression in RPE cells. K: Genetic inactivation of VEGF-A in myeloid cells does not prevent CNV lesion formation. L: CNV lesions exhibit no significant difference in neovascular area in mice that lack VEGF-A expression in myeloid cells, compared with age- and gender-matched lysozyme M Cre control mice. Data are expressed as means ± SD. n = 10 per group. Original magnification: ×10 (A, B, F, K); ×20 (C, D, E, J, K); ×40 (G).