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

¹From the Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan; and the ²Department of Pharmacology and the ³Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kwei-shan, Taoyuan, Taiwan.

PURPOSE. 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).

METHODS. 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.

RESULTS. 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.

CONCLUSIONS. 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.

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