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C. Cheng, C. Sun, H. Hsieh, C. Yang; Role of Proteolytic Enzymes in ex vivo Expansion of Human Limbal Epithelial Cells Cultivated on Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2007;48(13):454.
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Matrix breakdown is accomplished by several proteases including the PA/plasmin proteolytic axis and MMPs. In our previous study, we have demonstrated that limbal epithelial outgrowth on amniotic membrane (AM) is related to MMP-9 expression. We further investigate whether PA/plasmin system regulates MMP-9 expression associated with expansion of limbal epithelial cells on AM.
Corneoscleral buttons from human donor eyes were cut into 1.5 x 2 x 3 mm3 pieces and cultured on intact AM and plastic dishes in SHEM medium for 3 weeks. To determine the involvement of urokinase (uPA) in the outgrowth of limbal epithelial cells on AM, selective inhibitor of uPA (B428) and uPA receptor antibody were added at day 14 for another week. The extent of outgrowth was monitored with a phase contrast microscope and calculated by computer software. Activity and expression of MMP-9 were assessed by gelatin zymography, in situ zymography, RT-PCR, Western blotting, and immunofluorescence staining.
The activity and expression of uPA and MMP-9 were up-regulated in a time-dependent manner in H/AIntact and H group. The level of uPA and MMP-9 at 3rd week in H/AIntact group was higher than those in H group. MMP-9 enzymatic activity is co-localized with uPA activity by in situ zymography. To further confirm these results, treatment with uPA inhibitor B428 at day 14 attenuated the activity and expression of MMP-9 and cell outgrowth, as compared with control. Application of uPA receptor antibody decreases the expression and activity of MMP-9 on limbal epithelial cells cultured on AM, indicating that MMP-9 expression is regulated by PA/plasmin proteolytic enzymes in this model.
Our data suggested that PA/plasmin system is required for regulation of MMP-9 activity and expression, which modulate the interaction of cell-cell matrix in expansion of human limbal epithelial cells cultured on AM.
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