MMP-7 Knock-In Corneal Fibroblast Cell Lines Secrete MMP-7 with Proteolytic Activity towards Collagen XVIII

Abstract

Purpose To determine whether matrix metalloproteinase-7 (MMP-7) that is stably overexpressed by mouse corneal fibroblast cell lines exhibits proteolytic activity against the NC1 fragment of collagen XVIII. Methods Corneal fibroblasts isolated from MMP-7 knockout (7ko) mice were subjected to SV40 T-antigen immortalization and stably transfected with a bicistronic retroviral vector encoding green fluorescence protein and active MMP-7. The resulting MMP-7 knock-in fibroblasts (7ko-MMP-7 cells) were isolated and enriched by fluorescence activated cell sorting (FACS). Culture media samples from 7ko and 7ko-MMP-7 cells were then incubated with the recombinant NC1 fragment of collagen XVIII, and NC1 degradation was monitored by immunoblotting. Results Immunoblot analysis revealed that MMP-7 was present in lysates and culture media from 7ko-MMP-7 fibroblasts, but not media from immortalized 7ko fibroblasts. Importantly, lower amounts of the NC1 fragment were present in in vitro enzymatic reaction mixtures containing concentrated 7ko-MMP-7 media than in those containing concentrated 7ko media. Conclusion Immortalized fibroblasts stably transfected with MMP-7 secrete active MMP-7 with proteolytic activity towards the NC1 fragment of collagen XVIII.

Corneal Neovascularization and the Utility of Topical VEGF Inhibition: Ranibizumab (Lucentis) Vs Bevacizumab (Avastin)

“…176,177 This may be a function of the MMP-7-mediated cleavage of type XVIII collagen, the precursor of antiangiogenic endostatin. 167,178 …”

Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

“…These events were determined primarily using mouse corneas [9] , [10] . Although the use of a murine model to investigate corneal stromal cells has several advantages [1] , [5] , [11] , [12] , it is limited by the small amount of tissue available. Mouse corneas are small and thin and have an average diameter of 2.6 mm and thickness of 100 µm, two-thirds of which is stroma [13] , [14] .…”