Role of matrix metalloproteinase-9 in ex vivo expansion of human limbal epithelial cells cultured on human amniotic membrane.
OBJECTIVE
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.
Sun
Chi-Chin
CC
Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan.
Cheng
Ching-Yi
CY
Chien
Chin-Sung
CS
Pang
Jong-Hwei Su
JH
Ku
Wan-Chen
WC
Chen
Phil Yeong-Fong
PY
Yang
Chuen-Mao
CM
D000293:Adolescent; D000328:Adult; D000368:Aged; D000650:Amnion / Q000166:cytology*; D002465:Cell Movement; D002478:Cells, Cultured; D018920:Coculture Techniques; D004151:Dipeptides / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques; D016850:Limbus Corneae / Q000166:cytology* / Q000201:enzymology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000502:physiology*; D008875:Middle Aged; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D004151:Dipeptides; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D012333:RNA, Messenger; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9