Abstract
Purpose :
(i) To study the expression of pro-angiogenic MRC1+TIE2+ macrophages (TEMs) and Angiopoietin 2 (ANG2) in the normal vs. vascularized cornea. (ii) To test whether depletion of TEMs or inhibition of ANG2 is effective in reducing cornea neovascularization (CNV).
Methods :
FVB6-8 week mice were induced CNV with (i) alkali burn or (ii) suture placement and corneas were harvested at 6-10 days. Human avascular or vascularized corneas were also collected. Corneas were processed to fluorescence microscopy, immunohistochemistry, or flow cytometry. The following markers were studied: CD45, CD11b, CD11c, TIE2, MRC1, CD31, LYVE1, VWF, Collagen IV, and ANG2. FVB/TIE2-TK transgenic mouse bone marrow was transplanted into FVB mice, and selective suicide was induced into TIE2+ macrophages with topical Ganciclovir administration. FVB mice were treated with anti-ANG2 antibody systemically; two control groups received aspecific IgG and vehicle injection. We used a dulled blade to remove the basal epithelial membrane; a rotating burr to remove the epithelium + basal membrane. Macrophage infiltration, blood/lymphatic vessels, and ANG2 were quantified on whole mounts, with ImageJ software.
Results :
In the murine corneal stroma, all CD11b+ cells were also MRC1+TIE2+. In human corneas, 10.95±6.04% of MRC1+ cells was also TIE2+. There was a significant increase of stromal TEMs number following suture (p<0.001) or alkali burn (p<0.01) induced CNV. Following selective depletion of TEMs, hemangiogenesis (p<0.001) and lymphangiogenesis (p<0.05) were significantly reduced. Systemic administration of anti ANG2 antibody resulted in significant reduction of hemangiogenesis, lymphangiogenesis (p<0.001), and TEMs infiltration (p<0.0001).
Basal epithelial membrane disruption promoted pro-angiogenic TEMs infiltration (p<0.01), and ANG2 expression (p<0.001).
Conclusions :
The normal human and murine corneal (i) epithelium expresses high levels of ANG2; (ii) stroma is populated with pro-angiogenic macrophages (TEMs). Following CNV, TEMs infiltration and ANG2 expression are increased. Selective, in vivo ablation of TEMs, or pharmacological inhibition of ANG2, reduce hemangiogenesis and lymphangiogenesis. Finally, the basal epithelial membrane has a role in modulating ANG2 expression and pro-angiogenic TEMs infiltration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.