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D. Ma, J.–Y. Yao, J.–K. Chen, J.–S. Yu, K.–Y. Lin; Generation of Endostatin by Matrix Metalloproteinases and Cathepsins in Human Limbo–Corneal Epithelial Cells Cultivated on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4485.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously, we have shown that cultured human limbo–corneal epithelial cells (HLEC) secreted 21 and 26–28 kDa endostatin (EN)–related molecules, which were augmented when HLEC were cultivated on amniotic membrane. The purpose of this study is to identify proteolytic enzymes that generate EN from collagen XVIII in HLEC. Methods: HLEC were cultured on dish, intact (IAM) or denuded AM (DAM). Upon near confluence, inhibitors to MMPs (GM–6001, 1,10–Phenanthroline), cathepsins (E64, cathepsin B inhibitor II), elastase (Elastatinal), and serine proteases (AEBSF, aprotinin) were added individually or in combination to the cultures. Conditioned medium (CM) and cell lysate were collected. Samples were analyzed by gelatin zymography for gelatinase activity, and Western blot for cathepsins and EN. Results: Elastianal, AEBSF, and aprotinin had no effect on EN generation. MMP inhibitors inhibited the generation of both the 21 and 26–28 kDa EN, while cathepsin inhibitors inhibited predominantly the 21 kDa form. Combination of MMP and cathepsin inhibitors inhibited all forms of EN at lower concentrations. In addition, gelatin zymography revealed increased MMP–2/–9 activities especially in the CM of HLEC/DAM. Western blot analysis revealed increased cathepsin B but not cathepsin L in the CM of HLEC/IAM and HLEC/DAM. Conclusions: The result suggested that both the MMPs and cathepsins were involved in the generation of EN by HLEC, and a controlled increase in the proteolytic activity may be responsible for enhanced EN secretion by HLEC cultivated on AM.
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