Generation of endostatin by matrix metalloproteinase and cathepsin from human limbocorneal epithelial cells cultivated on amniotic membrane.
OBJECTIVE
Cultured human limbocorneal epithelial (HLE) cells secrete endostatin-related molecules that are augmented when the cells are cultivated on denuded amniotic membrane (DAM). This study is to identify mechanisms for enhanced endostatin production by HLE cells cultivated on AM.


METHODS
HLE cells were cultured on dish, on intact AM (IAM) or on DAM. Collagen XVIII alpha1 mRNA was analyzed by real-time quantitative PCR. In HLE/DAM cultures, inhibitors of MMPs (GM-6001; 1,10-phenanthroline), cathepsins (E64; cathepsin B inhibitor II), elastase (elastatinal), and serine proteases (AEBSF; aprotinin) were added. Endostatin in the conditioned medium (CM) was detected by Western blot. MMP-7; MMP-9; and cathepsins B, K, L, and V in the CM were quantitated by ELISA. Exogenous cathepsin B or V was added to the concentrated HLE/DAM CM to see the effect on endostatin production.


RESULTS
The expression of collagen XVIII alpha1 mRNA in the three groups was similar. Elastatinal, AEBSF, and aprotinin had no effect on endostatin generation. MMP inhibitors inhibited the generation of all the 20- and 28- to 30-kDa endostatin-related fragments, while cathepsin inhibitors inhibited only the 20-kDa endostatin. The level of MMP-7 and cathepsin B but not cathepsin V increased as the culture time increased, and paralleled with endostatin production. However, cathepsins K and L were absent in the CM. Exogenous cathepsins B and V further augmented the generation of endostatin.


CONCLUSIONS
MMP-7 and cathepsins B and V are involved in the generation of endostatin by HLE cells. Facilitating endostatin generation may be a novel physiological function of the cornea-specific cathepsin V.
Ma
David Hui-Kang
DH
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Kweishan Township, Taoyuan County 333, Taiwan. davidhkma@yahoo.com
Yao
Jeng-Yuan
JY
Kuo
Ming-Tse
MT
See
Lai-Chu
LC
Lin
Kuei-Ying
KY
Chen
Shao-Chun
SC
Chen
Jan-Kan
JK
Chao
An-Shine
AS
Wang
Su-Fang
SF
Lin
Ken-Kuo
KK
D000650:Amnion; D015153:Blotting, Western; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen / Q000235:genetics; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C509350:COL28A1 protein, human; D017077:Culture Media, Conditioned; D043169:Endostatins; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D012333:RNA, Messenger; D003094:Collagen; D002403:Cathepsins; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7