Abstract
The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions of the corneal and limbal epithelia in angiogenic and lymphangiogenic privilege, we designed five injury models involving debridement of different portions of the cornea and limbus and applied them to the dual-fluorescence reporter Prox1-GFP/Flt1-DsRed mouse, which permits in vivo imaging of blood and lymphatic vessels via fluorescence microscopy. Debridement of the whole cornea resulted in significant hemangiogenesis (HA) and lymphangiogenesis (LA), while that of the whole limbus yielded minimal corneal HA or LA. Following hemilimbal plus whole corneal debridement, corneal NV occurred only through the non-injured aspect of the limbus. Overall, these results suggest that the integrity of the corneal epithelium is important for (lymph)angiogenic privilege, whereas the limbus does not act as a physical or physiologic barrier to invading vessels. In CDh5-CreERT2VEGFR2lox/PGFD mice, conditional deletion of vascular endothelial growth factor receptor 2 in vascular endothelial cells abolished injury-induced HA and LA, demonstrating the utility of this transgenic mouse line for identifying important factors in the process of neovascularization.
Highlights
The cornea is a reliable model for studying the growth of blood and lymphatic vessels, due to its experimental accessibility and avascular nature
Endostatin, which imposes its antiangiogenic effects in the VEGF pathway and thereby halts endothelial cell proliferation, is produced from collagen type XVIII found in the extracellular matrix of the basement membrane[22]
PGFD mice circumvent the need for tissue preparation and the need to sacrifice mice between each observation point, allowing for the use of fewer mice and elimination of individual sample variations. This study demonstrates another utility of these mice—cross-breeding with a conditional vascular endothelial growth factor receptor 2 (VEGFR2) knockout model (CDh5-CreERT2VEGFR2lox/PGFD) to observe the importance of a well-studied receptor in vascular growth
Summary
The cornea is a reliable model for studying the growth of blood and lymphatic vessels, due to its experimental accessibility and avascular nature. To investigate the role of the limbus in corneal avascularity and the relative role of corneal factors in suppressing vascular invasion, we generated five distinct injury models that involve debridement of the epithelial layer of various regions, including the limbus, cornea, or both. We applied these models to our dual fluorescence reporter mice described previously (Prox1-GFP/Flt1-DsRed, or PGFD)[19]. We report the growth patterns of blood and lymphatic vessels in each of the five corneal and limbal injury models. We discuss the implications of our results with regards to the role of the limbus and the cornea in maintaining corneal avascularity
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