title: Generation of endostatin by matrix metalloproteinase and cathepsin from human limbocorneal epithelial cells cultivated on amniotic membrane.
abstract: 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.
authors.affiliation: Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Kweishan Township, Taoyuan County 333, Taiwan. davidhkma@yahoo.com
authors.forename: David Hui-Kang
authors.initials: DH
authors.lastname: Ma
authors.forename: Jeng-Yuan
authors.initials: JY
authors.lastname: Yao
authors.forename: Ming-Tse
authors.initials: MT
authors.lastname: Kuo
authors.forename: Lai-Chu
authors.initials: LC
authors.lastname: See
authors.forename: Kuei-Ying
authors.initials: KY
authors.lastname: Lin
authors.forename: Shao-Chun
authors.initials: SC
authors.lastname: Chen
authors.forename: Jan-Kan
authors.initials: JK
authors.lastname: Chen
authors.forename: An-Shine
authors.initials: AS
authors.lastname: Chao
authors.forename: Su-Fang
authors.initials: SF
authors.lastname: Wang
authors.forename: Ken-Kuo
authors.initials: KK
authors.lastname: Lin
mesh_terms: 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
chemical_list: 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