Abstract
Purpose::
To evaluate the role of endogenous Angiopoietin-2 (Ang2) in the development of corneal neovascularization and inflammation.
Methods::
Adult mice (7-14 weeks of age) homozygous for Ang2 deletion / LacZ substitution (Ang2-/-), their wild type (WT) littermates, and adult male C57Bl/6 mice were used in these studies. Corneal neovascularization (NV) was induced by intrastromal placement of 3 nylon sutures in the right eye (day 0). In C57Bl/6 mice, a neutralizing Ang2 antibody (25mg/kg) or a control protein (human Fc, 10mg/kg) was injected subcutaneously on days 0, 3 and 6 following corneal injury. On day 9 after corneal injury, the vasculature was labeled by intravenous injection of fluorescein conjugated lectin (lycopersicon esculentum), and corneal NV was evaluated in flat-mounts. Lymphatic vessels were co-localized in the same specimens by LYVE-1 immunostaining. Macrophage infiltration was evaluated in cross-sections stained with rat anti-mouse F4/80 monoclonal antibody. The Scion Image program was used for analysis of the area and length of corneal neovessels. Corneal thickness also was measured in cross-sections, as an index of edema.
Results::
The vasculature at the corneal limbus exhibited abnormalities in adult Ang2-/- mice, most notably a reduction in the caliber of the major circumferential veins and arteries. Following suture injury, corneal NV was significantly reduced in the Ang2-/- mice compared to their WT littermates (p <0.001) and also altered qualitatively, with the neovessels being much finer in the Ang2-/- mice. Genetic deletion of Ang2 also dramatically reduced the infiltration of macrophages into the damaged cornea. Systemic treatment with Ang2 antibody significantly inhibited corneal NV (p < 0.001) in C57Bl.6 mice. Ang2 antibody treatment also markedly decreased the growth of lymphatic vessels into the injured cornea and reduced corneal edema.
Conclusions::
These findings demonstrate that endogenous Ang2 plays an important role in the development of neovascularization, lymphangiogenesis and inflammation following corneal injury.
Keywords: growth factors/growth factor receptors • neovascularization • inflammation