Role of Matrix Metalloproteinase-9 in Ex Vivo Expansion of Human Limbal Epithelial Cells Cultured on Human Amniotic Membrane

Abstract

To investigate the expression and pivotal role of matrix metalloproteinase (MMP)-9 in the ex vivo expansion of human limbal explants with or without amniotic membrane (AM). Corneoscleral buttons were cultured on intact, denuded AM or plastic dishes for 3 weeks. To determine the role of MMP-9 in cell migration, either the MMP inhibitor GM6001 or an MMP-9 antibody was used. Expression of MMP-9 was determined by gelatin zymography, reverse transcription-polymerase chain reaction, and immunohistochemical staining. The expression of MMP-9 in all culture conditions increased in a time-dependent manner. However, the active form of MMP-9 emerged only in cultures on both intact and denuded AM from the second week. The averaged corrected ratio of MMP-9 expression in cultures on intact AM versus those on denuded AM or plastic dishes was 2.76 +/- 0.69- or 4.25 +/- 0.30-fold, respectively, when total RNA was used as an internal control. MMP-9 transcripts were upregulated in cultures on intact AM compared with the other two culture conditions. Immunohistochemical staining demonstrated that the MMP-9 protein was located on the limbal epithelial cells. Upregulation of MMP-9 associated with cell migration was significantly attenuated by both GM6001 and MMP-9 antibody, consistent with the inhibition of MMP-9 activity, as determined by gelatin zymography. In contrast, the sizes of limbal outgrowth were not different between the control and MMP-9 antibody-treated plastic dishes. These results demonstrated that MMP-9 not only was upregulated, it was also involved in the outgrowth of limbal epithelial cells. These results suggest that cell-cell matrix interaction is involved in the expansion of limbal epithelial cells on intact AM, and MMP-9 may be a key element.