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David Hui-Kang Ma, Jeng-Yuan Yao, Ming-Tse Kuo, Lai-Chu See, Kuei-Ying Lin, Shao-Chun Chen, Jan-Kan Chen, An-Shine Chao, Su-Fang Wang, Ken-Kuo Lin; Generation of Endostatin by Matrix Metalloproteinase and Cathepsin from Human Limbocorneal Epithelial Cells Cultivated on Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2007;48(2):644-651. doi: 10.1167/iovs.06-0884.
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purpose. 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 α1 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 α1 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.
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