Corneal neovascularization after excimer keratectomy wounds in matrilysin-deficient mice.
OBJECTIVE
Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing.


METHODS
Matrilysin-deficient mice (n = 17) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic factors, the levels of basic fibroblast growth factor (bFGF) were compared with those of vascular endothelial growth factor (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV).


RESULTS
The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% +/- 5.2% and 18.7% +/- 5.8% at days 3 and 7, respectively, compared with 5.3% +/- 2.4% and 5.5% +/- 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 +/- 0.55 mm(2) and 0.77 +/- 0.34 mm(2), respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV).


CONCLUSIONS
Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.
Kure
Tomoko
T
Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA.
Chang
Jin-Hong
JH
Kato
Takuji
T
Hernández-Quintela
Everardo
E
Ye
Hongqing
H
Lu
Paul Chung-Shien
PC
Matrisian
Lynn M
LM
Gatinel
Damien
D
Shapiro
Steven
S
Gosheh
Faris
F
Azar
Dimitri T
DT
Gosheh
Faris
F
D000818:Animals; D015153:Blotting, Western; D024141:Collagen Type IV / Q000378:metabolism; D003315:Cornea / Q000601:surgery*; D016510:Corneal Neovascularization / Q000201:enzymology* / Q000209:etiology; D003319:Corneal Stroma / Q000098:blood supply; D016228:Endothelial Growth Factors / Q000378:metabolism; D019573:Epithelium, Corneal / Q000201:enzymology / Q000473:pathology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D054018:Lasers, Excimer; D008222:Lymphokines / Q000378:metabolism; D008297:Male; D020783:Matrix Metalloproteinase 7 / Q000172:deficiency / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018903:Photorefractive Keratectomy / Q000009:adverse effects*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014945:Wound Healing
D024141:Collagen Type IV; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2; D020783:Matrix Metalloproteinase 7