title: Notch inhibition during corneal epithelial wound healing promotes migration.
abstract: OBJECTIVE
  To determine the role of Notch signaling in corneal epithelial migration and wound healing.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Asadolah
authors.initials: A
authors.lastname: Movahedan
authors.forename: Mercede
authors.initials: M
authors.lastname: Majdi
authors.forename: Neda
authors.initials: N
authors.lastname: Afsharkhamseh
authors.forename: Hossein M
authors.initials: HM
authors.lastname: Sagha
authors.forename: Niloufar S
authors.initials: NS
authors.lastname: Saadat
authors.forename: Keivan
authors.initials: K
authors.lastname: Shalileh
authors.forename: Behrad Y
authors.initials: BY
authors.lastname: Milani
authors.forename: Hongyu
authors.initials: H
authors.lastname: Ying
authors.forename: Ali R
authors.initials: AR
authors.lastname: Djalilian
mesh_terms: D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019573:Epithelium, Corneal / Q000293:injuries / Q000378:metabolism* / Q000473:pathology; D005131:Eye Injuries / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D012313:RNA / Q000235:genetics*; D051881:Receptor, Notch1 / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014945:Wound Healing / Q000235:genetics*
chemical_list: C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D012313:RNA