Pharmacologic uncoupling of angiogenesis and inflammation during initiation of pathological corneal neovascularization

Abstract

Pathological neovascularization occurs when a balance of pro- and anti-angiogenic factors is disrupted, accompanied by an amplifying inflammatory cascade. However, the interdependence of these responses and the mechanism triggering the initial angiogenic switch have remained unclear. We present data from an epithelial debridement model of corneal neovascularization describing an initial 3-day period when a substantial component of neovascular growth occurs. Administration of selective inhibitors shows that this initial growth requires signaling through VEGFR-2 (vascular endothelial growth factor receptor-2), independent of the accompanying inflammatory response. Instead, increased VEGF production is found prominently in repair epithelial cells and is increased prior to recruitment of neutrophil/granulocytes and macrophage/monocytes. Consequently, early granulocyte and monocyte depletion has little effect on corneal neovascularization outgrowth. These data indicate that it is possible to pharmacologically uncouple these mechanisms during early injury-driven neovascularization in the cornea and suggest that initial tissue responses are coordinated by repair epithelial cells.

FIGURE 1.

Epithelial debridement induces rapid and consistent corneal angiogenesis and inflammation by 3 days. A, representative FITC-concanavalin A-injected corneal flat mounts at various times over 20 days following epithelial debridement. B, quantification of total vascular area per cornea normalized to control (undebrided) limbal vessel areas (n = 10 corneas/group). Error bars indicate S.E. NV Area, neovascular area. C, H&E-stained corneal sections showing the progression of re-epithelialization over a matter of days. The images are all oriented with the limbus to the left (L) and the central cornea to the right (C). Note the repair epithelium at the wound edge (arrowhead) accompanied by underlying inflammatory infiltrates. By day 7, the cornea completely resurfaced with a stratified epithelium (E) but includes the presence of new shallow stromal vessels (S) (arrows). The presence of inflammatory cells was reduced by this time (20×).

FIGURE 2.

VEGFR-2 inhibition potently blocks corneal neovascularization and early angiogenesis markers in a dose-dependent manner. A, quantification of corneal neovascular areas (NV Area) after topical treatment with increasing doses of the VEGFR-2 antagonist, HGO452, resulting in a dose response with an ED₅₀ of 0.009% (n = 10, *, p < 0.05). B, CoNV areas after topical treatment with Dex, resulting in a dose response with an ED₅₀ of 0.1% drug (n = 10, *, p < 0.05). C, CoNV areas after treatment with 0.3% VEGFR-2 antagonists sorafenib or pazopanib showing significant inhibition as compared with vehicle (n = 8, *, p < 0.05). D, representative images from the experiment in A showing the potent inhibitory effects of 0.1% HGO452. E–H, corneal concentrations of vascular markers measured by multiplex ELISA over the initial 3 days of dosing for: control (no debridement), vehicle-treated, 1% Dex, or 0.1% HGO452. Concentrations of VCAM-1, ICAM-1, and VEGFR-2 itself were reduced for both Dex and HGO452. Concentrations of VEGF were reduced with Dex, but not HGO452 (n = 8). Error bars in panels A–C and E–H indicate S.E.

FIGURE 3.

Inhibition of VEGFR-2 signaling has minimal effects on early corneal inflammation. A, ELISA results from a corneal debridement time course for the neutrophil marker MPO showing reduced concentrations after treatment with 1% Dex, but not 0.1% HGO452, over 3 days following debridement (n = 8). B–F, multiplex ELISA results from corneal debridement time courses for proinflammatory cytokines MCP-1, IL-1β, TNF-α, TGF-β1, and the protease MMP-9 all displayed reduced concentrations after treatment with 1% Dex, but not 0.1% HGO452, over 3 days following debridement (n = 8). Error bars in all panels indicate S.E.

FIGURE 4.

Neutrophils and macrophages migrate into debrided cornea from limbus over several days and are not influenced by VEGFR-2 inhibition. A, representative corneal sections probed with the granulocyte marker, GR-1 (red), at various time points following debridement. Shallow stromal staining was notable by 6 h in the periphery and increased centrally with time, diminishing by day 7. This antibody also later recognized larger, macrophage-like cells in the stroma (e.g. day 3, central arrows) (40×). B, similar sections stained for the macrophage marker, F4/80, identifying cells throughout the corneal stroma that appear on day 2 and remain on day 7 (40×). C, GR-1-stained sections 48 h after debridement. Control (undebrided) sections showed no staining, vehicle-dosed sections showed prominent staining, and staining is reduced after treatment with 1% Dex, but remains robust after treatment with 0.1% HGO452 (10×). D, real-time RT-PCR results from corneas probed for the macrophage marker CSF1R and the neutrophil marker NCF1, and treated with vehicle, Dex, or HGO452. Increases in these markers were inhibited by Dex, but HGO452 had little effect. Error bars indicate S.E. Rel NCF1 Exp, relative NCF1 expression; Rel CSF1R Exp, relative CSF1R expression.

FIGURE 5.

VEGF expression increases prior to macrophage or neutrophil recruitment and is localized to repair epithelium. A, quantitative RT-PCRs from debrided corneas over 6 days probed for VEGF, the macrophage marker CSF1R, and the neutrophil marker NCF1. VEGF expression sharply peaked by 6 h, whereas CSF1R slowly increased over 3 days, and NCF1 peaked at 24 h (n = 4). Rel VEGF Exp, relative VEGF expression; Rel NCF1 Exp, relative NCF1 expression; Rel CSF1R Exp, relative CSF1R expression. B, relative integrated density (Rel Int Dens) of VEGF staining quantified from the 100-μm wound edge epithelial (Epith) cells showed an increase as compared with adjacent epithelial regions on the same section from days 1–3 (n = 4, *, p < 0.05). Error bars in panels A and B indicate S.E. C, representative images from B. Prominent epithelial staining for VEGF protein was present in peripheral repair epithelium and weak in keratocytes and endothelial cells on day 2. Staining was consistently more intense in wound edge epithelial cells from the same section (arrowhead, 20×). VEGF staining was absent or very weak in inflammatory cells at this time (arrows) (20×). EP, epithelium; ST, stroma; EN, endothelium. D, staining for the corneal epithelial keratin 12 (K12) is strongly present in control (naive) corneas, but not in the resurfaced repair epithelium at 7 days following debridement (10×).

FIGURE 6.

Early depletion of neutrophils/granulocytes and macrophages/monocytes does not inhibit corneal angiogenesis following debridement. A, blood neutrophil counts following corneal debridement after administration of three serial doses of GR-1. Neutrophils were strongly inhibited at 30 h as compared with IgG control, and this trend was maintained to day 6 (n = 4, *, p < 0.05). B, blood monocyte counts from the same samples. Monocytes were also strongly inhibited at 30 h as compared with IgG control (n = 4, *, p < 0.05). C, corneal MPO concentrations 30 h after debridement were significantly reduced as compared with IgG control (n = 8, *, p < 0.05). D, quantification of neovascular area (NV Area) showed no significant difference between GR-1- and IgG-injected groups (n = 8). Error bars in panels A–D indicate S.E. E, representative images from D showing substantial neovascular growth in GR-1 and control corneas by day 6.

FIGURE 7.

Model of CoNV initiation. Repair epithelial cells (Repair Epith) at the wound edge secrete pro-inflammatory cytokines, including VEGF, leading to angiogenesis from limbal vessels and recruitment of neutrophils (Neuts) and macrophages (Macs). These processes are initially separate, but may become interdependent over time.