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Novel Laminin 5 2-chain Fragments Potentiating the Limbal Epithelial Cell Outgrowth on Amniotic Membrane

¹From the Departments of Physiology, ²Pharmacology, and ⁵Chinese Medicine, and the ⁹Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan; the ⁴Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan; the ⁶Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan; the ⁷Department of Nursing, Division of Basic Medical Sciences, Chang Gung Institute of Technology, Tao-Yuan, Taiwan; and the ⁸Department of Ophthalmology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan.

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 2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 2-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 2-chain to facilitate limbal outgrowth on intact AM.