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C.–C. Sun, C.–Y. Cheng, H.–F. Cheng, J.–H.S. Pang, W.–C. Ku, P.Y. Chen, J.–K. Chen, C.–C. Chen, C.–M. Yang; Phosphatidylinositol 3–Kinase/Akt, but Not p42/p44 MAPK Pathway Regulates Matrix Metalloproteinase–9 Expression in ex vivo Expansion of Human Limbal Epithelial Progenitor Cells on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1611.
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© ARVO (1962-2015); The Authors (2016-present)
Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) are capable of restoring the ocular surface with limbal stem cell deficiency (LSCD). Previously, we have demonstrated the role of matrix metalloproteinase–9 (MMP–9) in the outgrowth of expanded human limbal epithelial cells on intact AM. In the present study, we sought to elucidate the signaling pathways mediating MMP–9 expression in this model.
Corneoscleral buttons from human donor eyes were cut into 1.5 x 2 x 3 mm3 pieces and cultured on intact AM in SHEM medium for 3 weeks. When addition of pharmacological inhibitors was necessary to detect their effects on the outgrowth of limbal epithelial cells, different concentrations of inhibitors of phosphatidylinositol 3–kinase (PI3–K), NFΚB (nuclear factorΚB), p38 and p42/p44 MAPK were added from day 14 for another week. The extent of each outgrowth was monitored with a phase contrast microscope and calculated by computer software. Expression and regulation of MMP–9 during such expansion was studied by gelatin zymography, in situ zymography, reverse transcription–polymerase chain reaction (RT–PCR), and immunohistochemical staining.
MMP–9 was preferentially expressed at the leading edge of limbal epithelial outgrowth. LY294002 as well as Wortmannin at 30 µM (PI3–K inhibitors), Helenalin at 10µM (NFΚB inhibitor) and U0126 at 10 µM (p42/p44 MAPK inhibitor) significantly suppressed the expansion of limbal epithelial cells on intact AM as compared to the control group, whereas the outgrowth was not inhibited by 30 µM of SB202190 (MAPK p38 inhibitor). However, gelatin zymography demonstrated that enzymatic activity of MMP–9 was attenuated by LY294002, Wortmannin and Helenalin, but not affected by U0126 and SB202190, which was confirmed by the results of RT–PCR. Furthermore, LY294002 also down–regulated phosphorylated–Akt expression as well as NFΚB nuclear translocation at the migration leading edge as evidenced by confocal microscopy.
PI3–K/Akt/NFΚB, but not p42/p44 MAPK pathway regulates MMP–9 expression in ex vivo expansion of human limbal epithelial progenitor cells on intact AM.
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