September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Macrophages, angiopoietin 2, and corneal neovascularization: translational implications
Author Affiliations & Notes
  • Giulio Ferrari
    San Raffaele Scientific Institute, Milan, Italy
  • chiara giacomini
    San Raffaele Scientific Institute, Milan, Italy
  • Fabio Bignami
    San Raffaele Scientific Institute, Milan, Italy
  • davide moi
    San Raffaele Scientific Institute, Milan, Italy
  • anna ranghetti
    San Raffaele Scientific Institute, Milan, Italy
  • claudio doglioni
    San Raffaele Scientific Institute, Milan, Italy
  • luigi naldini
    San Raffaele Scientific Institute, Milan, Italy
  • Paolo Rama
    San Raffaele Scientific Institute, Milan, Italy
  • roberta mazzieri
    Diamantina Institute Faculty of Medicine and Biomedical Sciences, The University of Queensland , Brisbane, Queensland, Australia
  • Footnotes
    Commercial Relationships   Giulio Ferrari, None; chiara giacomini, None; Fabio Bignami, None; davide moi, None; anna ranghetti, None; claudio doglioni, None; luigi naldini, None; Paolo Rama, None; roberta mazzieri, None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3529. doi:
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      Giulio Ferrari, chiara giacomini, Fabio Bignami, davide moi, anna ranghetti, claudio doglioni, luigi naldini, Paolo Rama, roberta mazzieri; Macrophages, angiopoietin 2, and corneal neovascularization: translational implications. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3529.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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