April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Sheets of Corneal Epithelial Cells Detect Edges
Author Affiliations & Notes
  • J. K. Klarlund
    Ophthal & Vis Sci Res Ctr, Univ of Pittsburgh Eye & Ear Inst, Pittsburgh, Pennsylvania
  • E. R. Block
    Ophthal & Vis Sci Res Ctr, Univ of Pittsburgh Eye & Ear Inst, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  J.K. Klarlund, None; E.R. Block, None.
  • Footnotes
    Support  This work was supported by the National Institutes of Health Grants EY013463 and EY08098 and grants from Research to Prevent Blindness and The Eye and Ear Foundation (Pittsburgh, PA).
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3497. doi:
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      J. K. Klarlund, E. R. Block; Sheets of Corneal Epithelial Cells Detect Edges. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3497.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Activation of the EGF receptor is a key event in inducing the corneal epithelium to migrate and cover wounds. When wounds are inflicted in an epithelium, edges are created. We sought to determine whether the presence of edges is in itself a trigger for activation of the EGF receptor.

Methods: : Cells were cultured on thin plastic strips: Tissue culture plates were covered with poly(2-hydroxyethyl methacrylate) (poly-HEMA) which effectively blocks cell attachment, thin strips of plastic dissolved in chloroform were applied, dried, and HCLE (immortalized human corneal limbal epithelial) cells seeded by conventional procedures. As controls, cells were seeded on poly-HEMA plates that were totally covered with plastic. Activated EGF receptor and ERK1/2 were detected by blotting with phospho-specific antibodies. Cell division was monitored by thymidine incorporation, and by blotting with the Ki67 antibody.

Results: : We have constructed a tissue culture system that allows analysis of signaling in cultures of confluent epithelial cells that contain many free edges versus cultures containing few edges. Confocal microscopy confirmed that cells at the edges of the strips were physically unconstrained. The cells exhibited comparable density-dependent inhibition of growth under the two conditions, and had a similar epithelial morphology and distributions of f-actin. Strong stimulation of ERK1/2 was observed in cultures containing many edges, which was dependent on EGF receptor signaling. The EGF receptor was downregulated, as is expected after prolonged activation. The low levels of ATP in the supernatnants and experiments with apyrase suggested that activation of the EGF receptor was independent of purinergic signaling. The presence of edges induced much lower levels of production of MMP9 than did induction of wounds in the cell sheet by more conventional means, suggesting that other stimuli, for example integrin engagement to denuded extracellular matrix, promotes production of MMP9.

Conclusions: : The very presence of edges in a sheet of epithelial cells is sufficient to induce activation of the EGF receptor signaling pathway, which is central to promoting motility.

Keywords: cornea: epithelium • wound healing • cornea: basic science 

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