VEGF-dependent conjunctivalization of the corneal surface.
OBJECTIVE
To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency.


METHODS
The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization.


RESULTS
Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001).


CONCLUSIONS
Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.
Joussen
Antonia M
AM
Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA.
Poulaki
Vassiliki
V
Mitsiades
Nicholas
N
Stechschulte
Stephen U
SU
Kirchhof
Bernd
B
Dartt
Darlene A
DA
Fong
Guo-Hua
GH
Rudge
John
J
Wiegand
Stanley J
SJ
Yancopoulos
George D
GD
Adamis
Anthony P
AP
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002452:Cell Count; D003228:Conjunctiva / Q000378:metabolism* / Q000473:pathology; D016510:Corneal Neovascularization / Q000378:metabolism* / Q000473:pathology; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004847:Epithelial Cells / Q000473:pathology; D020397:Goblet Cells / Q000473:pathology*; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008222:Lymphokines / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase / Q000378:metabolism
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D040281:Vascular Endothelial Growth Factor Receptor-1; D001616:beta-Galactosidase