Abstract
To determine the role of Notch signaling in corneal epithelial migration and wound healing. Immunolocalization of Notch1 was performed during epithelial wound healing in vivo in mouse corneal epithelial debridement wounds and in vitro in primary human corneal epithelial cells following a linear scratch wound. The effects of Notch inhibition, using the γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or following stable transfection with Notch1-short hairpin RNA (shRNA), was evaluated in a scratch assay and transwell migration assay. Likewise, in vitro adhesion, proliferation and the actin cytoskeleton was examined. The DAPT effect was also evaluated in vivo in a mouse model of corneal epithelial wound healing. The expression of Notch1 was reduced at the leading edge of a healing corneal epithelium both in vivo and in vitro. Notch inhibition using DAPT and using Notch1-shRNA both enhanced in vitro migration in scratch and transwell migration assays. Consistent with this increased migratory behavior, Notch inhibited cells demonstrated decreased cell-matrix adhesion and enhanced lamellipodia formation. Notch inhibition by DAPT was also found to accelerate corneal epithelial wound closure in an in vivo murine model without affecting proliferation. The results highlight the role of Notch in regulating corneal epithelial migration and wound healing. In particular, Notch signaling appears to decrease in the early stages of wound healing which contributes to cytoskeletal changes with subsequent augmentation of migratory behavior.
Highlights
The results highlight the role of Notch in regulating corneal epithelial migration and wound healing
The corneal epithelium protects the cornea against pathogen invasion and is essential for maintaining the integrity and clarity of the cornea
This highly coordinated process involves a number of cellular functions including migration, proliferation, and differentiation, which in many ways recapitulate the same pathways involved during development
Summary
Immunolocalization of Notch[1] was performed during epithelial wound healing in vivo in mouse corneal epithelial debridement wounds and in vitro in primary human corneal epithelial cells following a linear scratch wound. The effects of Notch inhibition, using the c-secretase inhibitor N-(N[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or following stable transfection with Notch1-short hairpin RNA (shRNA), was evaluated in a scratch assay and transwell migration assay. The DAPT effect was evaluated in vivo in a mouse model of corneal epithelial wound healing. Human corneal epithelial cell cultures were initiated from cadaver corneas and kindly provided by the Illinois Eye Bank. Cells were washed and resuspended in keratinocyte serum free medium (KSFM; Invitrogen, Grand Island, NY) and plated in collagen coated tissue culture plates. HCE-T cells were grown in Dulbelcco’s modified Eagle’s medium (DMEM)/F12 (1:1) with 5% fetal bovine serum (FBS).
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call