Role of Non-Endothelial Cells in Corneal Angiogenesis

Abstract

Purpose: It has been suggested that growth of blood and lymphatic vessels in the cornea is assisted by non-endothelial cells in the stroma, in particular, during later stages when a vascular network is established by anastomosis. Supporting roles of stromal myeloid cells and an existing nerve network have been suggested, but molecular and cellular mechanisms remain unresolved. The aim of this study is to examine evidence for the participation of stromal, non-endothelial cells during the vascular network formation.

Methods: For tests of myeloid cells, four corneal angiogenesis models in the mouse eye were used. 1) suture placement, 2) topical application of ML9 (MLCK inhibitor), 3) Dstn^corn1 mouse (spontaneous corneal vascularization model), 4) corneal conjunctivalization following total epithelial removal. Blood vessel growth was monitored in a live eye with fluorescence angiography. Cell distribution was determined by whole-mount immunofluorescence with CD11b, CD45, CD90.2, F4/80, MHC-class II, CD31 (blood vessels) and LYVE1 (lymphatic vessels). Cell distribution was quantified in an area of vascular growth front where a vascular network is formed by anastomosis. For a nerve guidance test, suture was placed in the cornea of Thy1-CFP mice, and both blood vessels (fluorescence angiography) and nerves (CFP) were tracked by time-lapse imaging in live animals.

Results: Untouched, avascular corneas contained a small number of F4/80, CD11b and MHC-class II positive cells, primarily in the limbal area and sparsely throughout the entire cornea. CD90.2 positive cell distribution had no limbal preference but was closely associated with sub-basal nerve plexus. After initiation of experimental angiogenesis, the number of cells with one or more of myeloid markers greatly increased within an area of vascular growth. There were frequent instances of a F4/80 positive cell being present between two sprouting tips of growing vessels, appearing to bridge them. Time-lapse imaging revealed no clear correlation between vessel growth and a CFP-positive nerve fiber network.

Conclusions: Myeloid cells may play a supporting role in network formation of pathological corneal vasculature either in a paracrine manner or by means of fusion with endothelial tip cells, as reported in developing brain. A guiding role of an existing nerve network, on the other hand, is not supported by the data.

Keywords: 609 neovascularization • 554 immunohistochemistry • 480 cornea: basic science