Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.The cornea is typically avascular in its normal state. However, corneal neovascularization (NV) occurs in conjunction with several corneal diseases such as infection, injury, and autoimmune reactions and is one of the leading causes of blindness. Recent studies have identified several tyrosine kinases and their corresponding ligands that mediate NV, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).^1,2,3bFGF was first identified as a pro-angiogenic factor and is studied extensively in corneal NV models because it is thought to be a major factor in the induction of corneal NV.^4,5,6 bFGF is secreted by corneal epithelial cells, stromal fibroblasts, and endothelial cells, and is localized to the corneal extracellular matrix.⁷ Low levels of bFGF are produced in unwounded corneas; however, enhanced bFGF production was detected in corneal epithelial cells after injury.⁸ VEGF was also shown to promote NV in corneal wounding models,⁹ and cross talk is thought to occur between bFGF and VEGF during corneal NV. For example, bFGF was shown to induce corneal NV by activating the VEGF/VEGFR system^10,11 and the systemic administration of anti-VEGF-A neutralizing antibodies dramatically reduces this effect.¹²Membrane type-1 matrix metalloproteinase (MT1-MMP) is the first transmembrane-containing matrix metalloproteinase to be identified.¹³ Based on previous reports using corneal wound-healing models, MT1-MMP mRNA is mainly localized to the corneal stroma.¹⁴ During NV, quiescent endothelial cells are activated and migration is facilitated by degrading the extracellular matrix through the action of specific proteases, including MT1-MMP.^15,16,17 The importance of the enzymatic function of MT1-MMP in corneal NV was shown using the corneal pocket assay in MT1-MMP-deficient mice.¹⁸ Interestingly, the expression of MT1-MMP is up-regulated by bFGF stimulation in prostate carcinoma cell lines,¹⁹ and it was also reported that MT1-MMP promotes VEGF secretion.^{20,21,22,23,24,25}In this study, we developed anti-MT1-MMP antibody to localize and characterize MT1-MMP protein in the mouse cornea. To assess the relationship between MT1-MMP and bFGF during corneal NV, we performed experiments that combined the corneal pocket assay using a bFGF pellet with the injection of naked MT1-MMP DNA. We observed an enhanced phosphorylation of MAP kinases in wild-type and MT1-MMP knockin (KI) cell lines over that of MT1-MMP knockout (KO) cell lines, suggesting a role of MT1-MMP in modulating bFGF-mediated signal transduction pathways.