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T. Kawakita, E.M. Espana, W. Li, H. He, S.C. G. Tseng; Epithelial–Mesenchymal Transition by Limbal Epithelial Progenitor Cells Leads to Intrastromal Invasion, Limbal Stem Cell Deficiency and Fibrosis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3500.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To elucidate whether the cellular mechanism of l epithelia–mesenchymal transition (EMT) in involved in imbal stem cell (SC) deficiency. Methods: Corneo–limbo–conjunctival rings from both pigmented Dutch belt and New Zealand albino rabbits were subdivided into 6 explants, placed on a polyester membrane and cultured in SHEM by a submerged manner or at the air–fluid interface (AL) for 2, 4 and 7 days. Isolated limbal and corneal epithelial sheets were recombined with live or dead limbal stroma and cultured under AL for 7 days. Epithelial outgrowth, limbal pigment line migration, and epithelial intrastromal invasion were assessed by histology and by immunostaining to p63, Ki67, E–cadherin, ß–catenin, pancytokeratins and keratin 3. Surface epithelial cells removed by dispase and cells released by collagenase from the remaining stroma of day 7 submerged or air–lifted explants were cultured on plastic for 24 h or on 3T3 fibroblast feeder layers for 10 days, and immunostained by antibodies against pancytokeratins, p63 and S100A4. Results: As compared to the normal or submerged control, AL explants showed significantly more epithelial migration, stratification and outgrowth. Furthermore, AL, but not submerged, explants showed pronounced intrastromal invasion by K3–expressing cells in the conjunctival region and digital invasion into the limbal stroma by limbal basal progenitor cells. The latter phenomenon was accompanied by upregulated nuclear expression of p63 and Ki67, downregulated E–cadherin and cornea–specific keratin 3, and switched expression of ß–catenin from intercellular junctions to the nucleus and cytoplasm, collectively indicating activation of Wnt/ß–catenin pathway. Furthermore, intrastromal invasion took place by limbal, but not corneal, epithelial sheets when combined with live, but not dead, limbal stroma. Invaded cells isolated by collagenase from the stroma of AL, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete EMT into fibroblasts expressing nuclear p63 and cytoplasmic S100A4. Conclusions: These findings support a new paradigm that EMT via Wnt/ß–catenin pathway influences the fate decision of limbal epithelial SC between regeneration and fibrosis when the stromal niche is activated by air–lifting to simulate the wound healing process. This model can help dissect the pathogenesis of limbal SC deficiency under inflammation.
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