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Ching-Yi Cheng, Chi-Chin Sun, Wei-Hsuan Yu, Hsi-Lung Hsieh, David Hui-Kang Ma, Jong-Hwei Su Pang, Chuen-Mao Yang; Novel Laminin 5 γ2-chain Fragments Potentiating the Limbal Epithelial Cell Outgrowth on Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2009;50(10):4631-4639. doi: 10.1167/iovs.08-3060.
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purpose. Matrix metalloproteases (MMPs)-mediated extracellular matrix (ECM) degradation potentially releases cryptic motility factors involved in somatic stem cell migration and epithelial outgrowth. The authors previously demonstrated that MMP-9 is upregulated in limbal epithelial cells cultivated on amniotic membrane (AM). Here, the authors further investigated whether plasminogen activator (PA)/plasmin regulates MMP-9 activity in this model implicated in the processing of laminin 5 (Ln5), a component of amniotic basement membrane.
methods. Limbal epithelial cells migrated from limbal explants were expanded on intact AM. The activities and proteins of uPA and MMP-9 in limbal epithelial cells were determined by fibrin and gelatin zymography, reverse transcription–polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Specific pharmacological inhibitors including MMPs inhibitor GM6001, MMP-2/-9 inhibitor, and uPA inhibitor B428 were used to determine whether the PA/plasmin/MMP-9 axis induces cell growth via Ln5 in this model.
results. These data showed that MMP-9 activity was attenuated by a selective uPA inhibitor, B428. Furthermore, MMP-9 activity was enhanced by exogenous addition or pre-incubation with plasmin. These results demonstrated that PA/plasmin regulates MMP-9 expression. An interesting proteolytic fragment of Ln5 γ2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 γ2-chain is processed by uPA/MMP-9. Moreover, the extent of limbal outgrowth was also retarded by B428.
conclusions. This study suggested that MMP-9 activity was upregulated by PA/plasmin, which in turn processed Ln5 γ2-chain to facilitate limbal outgrowth on intact AM.
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