Effect of MT1–MMP Knockout and Knock–In Corneal Epithelial Cells and Keratocytes on Vascular Endothelial Cell Migration and Proliferation

Abstract

Abstract: : Purpose: Previous studies from our laboratory have shown that MT1–MMP induces neovascularization associated with increased VEGF production in vivo. The purpose of this study was to investigate the influence of MT1–MMP on vascular endothelial cell migration and proliferation using MT1–MMP knockout (KO) and knock–in corneal epithelial and keratocyte cell lines. Methods: Immortalized corneal cells from WT and MT1–MMP KO mice were subcloned to generate corneal epithelial and keratocyte cell lines (4 cell lines characterized immunohistochemically using anti–keratin AE1/AE3, anti–vimentin, anti–integrin α5ß1, and anti–α smooth muscle actin). They were stably transfected with MT1–MMP, empty vector, and two MT1–MMP mutants (TC and E240), followed by fluorescence activated cell sorting, generating 14 additional cell lines. For the Boyden chamber migration assays, 440 µl/well of supernatant from the cell lines were used as chemoattractant in the lower chamber, and 250,000 CPAE cells/well in the upper chamber. For the proliferation assays, 15,000 CPAE cells/well were seeded in a 96–well plate, starved for 24 hours, and subsequently replaced by supernatant from cell lines. Forty–eight hours later, colored formazan product assay was used to calculate the number of living CPAE cells. Normalized data were compared using two–tailed Z–test. Results: Epithelial and keratocyte MT1–MMP KO cell lines and 10% FCS resulted in significantly increased CPAE cells proliferation (186.7±2.1%, 190.5±6.0%, and 188.0±4.6%, respectively) as compared to baseline (100%, CPAE cells in 0.5% FCS). These were significantly higher than WT and KO MT1–MMP transfected corneal epithelial and keratocyte cell lines, except for KO MT1–MMP knocked in MT1–MMP–TC (176.5±1.8%) among transfected corneal epithelial cell lines, and for KO MT1–MMP knocked in MT1–MMP (184.2±5.7%) among transfected keratocyte cell lines. Migration assays confirmed these findings. Conclusions: Despite increased VEGF production associated with MT1–MMP transfection in vivo, overexpression of transfected MT1–MMP in keratocyte and corneal epithelial cells reduces vascular endothelial cell migration and proliferation in vitro.

Keywords: neovascularization • cornea: basic science • gene/expression