Role of Keratocan in Corneal Epithelium During Wound Healing

Abstract

Abstract: : Purpose: Keratocan has been defined as structural extracellualr keratan sulfate proteoglycan (KSPG) in the normal cornea. In this study, we investigate the role of keratocan in corneal wound healing Methods: Keratocan–null (Kera^–/–) mice and wild–type littermate were employed in epithelium debridement wound healing experiments. The central cornea was marked by a trephine of 2 mm in diameter and the epithelium was debrided by an Algerbrush IITM corneal rust ring remover with a 0.5 mm burr under a stereo–microscope. The corneal wound healing was evaluated both in vivo and in vitro in an organ culture system. The extent of corneal wound closure was examined at 6∼48 hr after debridement by fluorescein staining and photographed using Axiovision4 digital camera (ZEISS). The animals were sacrificed and the eyeballs were enucleated for histological and biochemical analysis. Keratocan expression was detected by western blotting and immunohistochemistry with an epitope–specific anti–mouse keratocan antibody Results: Corneal epithelial wound healing experiment showed that most of corneal epithelium healed at 48 hr post–debridement in the wild–type mouse corneas and less than 20% remain small punctuate defect, while 80% of the Kera^–/– cornea still remain defect at 48 hr post–debridement. Interestingly, keratocan, expressed only by the keratocyte in normal cornea, was indeed transiently and ectopically detected by the corneal epithelial cells during wound healing. Western blotting analysis showed that keratocan exists as proteoglycan form in the unwounded cornea, but as glycoprotein form in the healing epithelial cells. .Adding anti–keratocan antibody at 50 µg/ml in an organ culture system was able to retard the rate of corneal epithelial wound healing. Conclusions: These results indicated that in addition to being a structural extracelluar matrix protein, keratocan may actively involve in facilitating corneal epithelial wound healing.

Keywords: cornea: epithelium • wound healing • proteoglycans/glycosaminoglycans