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F. Bock, J. Onderka, T. Dietrich, B. Bachmann, F. Kruse, C. Cursiefen; Aqueous Humour Displays Antilymphangiogenic Effects. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3200. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The normal cornea is devoid of both blood and lymphatic vessels. It is unknown how the normal cornea prevents ingrowths of lymphatic vessels from the limbus. Purpose of this study was to analyze, whether aqueous humor (AH), constituting a fluid compartment close to the cornea, affects lymphangiogenesis.
A potential inhibitory effect of AH was first analyzed in vitro by quantifying lymphatic endothelial cell (LEC) proliferation with a colorimetric (BrdU) proliferation ELISA. LECs were seeded in a 96-well plate in EGM2-MV medium at a density of 2 x 103 cells/well and left over night to attach. Medium was replaced with serum-, bFGF- and VEGF-A-free EGM2-MV medium (minimal medium). 5 or 20 µl of human AH and BrdU were added. As control, equal volumes of minimal medium were added instead of AH. Cells were fixed and stained after 3 days according to manufactures' instructions. Secondly, in the murine model of inflammatory corneal neovascularization the potential antilymphangiogenic effects of AH were analyzed in vivo. Three interrupted 11-0 nylon sutures were placed into the cornea (BALB/c mice) and left in place for five days. The treatment group (n=5) received 3 µl of murine AH once a day at day of surgery and for the following three days. Control mice received an equal volume of saline solution. After five days, mice were sacrificed and corneal whole mounts prepared. For immunohistochemistry, corneal flat mounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and CD31 as panendothelial marker. Morphometry of blood and lymphatic vessels was performed with the image analysis software analySIS^B.
Human AH inhibited the proliferation of human LECs in vitro dose dependently (5 µl: p>0.0847 n.s; 20 µl: p< 0.0001). The topical application of murine AH in vivo inhibited the outgrowth of both blood (p < 0.006) and lymphatic vessels significantly (p< 0.0002).
Aqueous humor inhibits the proliferation of lymphatic endothelial cells in vitro and the outgrowth of lymphatic vessels in the cornea in vivo. Aqueous humour therefore seems to play an important role in maintaining corneal lymphangiogenic privilege.
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