Abstract
Purpose: :
CAP37, a cationic antimicrobial protein, mediates proliferation, migration, and adhesion of human corneal epithelial cells (HCEC), which are essential processes of corneal wound healing. The purpose of these studies was to test the hypothesis that CAP37 facilitates the healing of corneal wounds in vitro and in vivo through the protein kinase C (PKC) signaling pathway.
Methods: :
Confluent HCEC monolayers were "wounded" in a perpendicular fashion utilizing a 10μl pipette tip followed by the addition of recombinant CAP37 (rCAP37, 25-2000ng/ml), basal media, and heparin-binding epidermal growth factor (HB-EGF, 250ng/ml). Wound closure was recorded (0, 24, and 48h) utilizing a camera on an inverted microscope. Immunocytochemistry was performed on treated and untreated scratched HCEC monolayers for the presence of PKC Δ. In an in vivo model of corneal wound healing, a 2mm wound was created utilizing the Algerbrush II. Wounds were treated with rCAP37 (250ng/ml), HB-EGF (50ng/ml), or vehicle control at 16 and 24h. Wound closure was monitored via fluorescein staining, measured, and quantitated using ImageJ software. The healing process was further analyzed by histology.
Results: :
Corneal wound healing studies revealed that rCAP37 significantly increased wound closure in vitro (P<0.01) and in vivo (P<0.05) compared to untreated wounds. In vitro results showed that rCAP37 maximally facilitated wound closure in a dose responsive manner between 250 and 500ng/ml. Confirmation of in vitro data were obtained in vivo showing an increased percentage in wound closure in wounds treated with 250ng/ml rCAP37 over vehicle treated wounds. Histology revealed neutrophil infiltration at 16h in rCAP37 and HB-EGF treated wounds. Re-epithelialization of the corneal epithelium in rCAP37 and HB-EGF treated wounds was complete by 24h. Immunocytochemistry of in vitro wounds revealed the presence of PKC Δ along the wound edge with increased amount of staining for PKC Δ in rCAP37-treated monolayers when compared to untreated monolayers.
Conclusions: :
These data strongly confirm that CAP37 facilitates corneal wound healing and mediates healing through the PKC pathway, specifically though PKC Δ. These findings contribute to the development of CAP37 as a therapeutic for corneal injuries.
Keywords: wound healing • cornea: epithelium • inflammation