Abstract

PURPOSE. Fibronectin plays an important role in the migration of corneal epithelial cells in vivo. The Arg-Gly-Asp (RGD) sequence in the principal cell binding domain of fibronectin mediates the interaction of fibronectin with integrins, whereas the Pro-His-Ser-Arg-Asn (PHSRN) sequence of fibronectin is thought to modulate this interaction. The authors examined the effects of a PHSRN peptide on corneal epithelial migration in vitro and in vivo. METHODS. Epithelial migration in vitro was examined with the rabbit cornea in organ culture. The motility and phenotype of simian virus 40 -transformed human corneal epithelial (HCE) cells were evaluated by time-lapse and immunofluorescence microscopy, respectively. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was examined by immunoprecipitation and immunoblot analysis. The healing of rabbit corneal epithelial wounds induced by 1-heptanol was evaluated by fluorescein staining. RESULTS. The PHSRN peptide stimulated corneal epithelial migration in organ culture in a concentration-dependent manner, and it increased HCE cell motility in vitro. The peptide induced the accumulation of F-actin and the formation of focal adhesions at the leading edge of HCE cells. It also upregulated the tyrosine phosphorylation of FAK and paxillin in HCE cells, but it did not affect HCE cell proliferation or attachment to a fibronectin matrix. Administration of the PHSRN peptide in eye drops promoted corneal epithelial wound closure in vivo in a dose-dependent manner. None of these effects of the PHSRN peptide were induced by a control NRSHP peptide. T he healing of corneal epithelial wounds is important for maintaining corneal transparency. Damage to the corneal epithelium induces migration of the remaining epithelial cells to cover the area of the defect. Thus, the motility of corneal epithelial cells is key to the initiation of wound healing. We and others 1,2 have shown that fibronectin, an adhesive extracellular glycoprotein, appears at the exposed surface of the stroma at corneal epithelial wound sites. Epithelial cells migrate over this provisional matrix of fibronectin and upregulate their expression of integrin chains that form the cell surface receptors for fibronectin. CONCLUSIONS. 3 The fibronectin-integrin system thus plays a central role in corneal epithelial wound healing. 5 Furthermore, a fibronectin matrix promotes the attachment of corneal epithelial cells in culture, and this effect is inhibited by the addition of an RGD (Arg-Gly-Asp) peptide corresponding to the principal cell-binding domain of fibronectin (Gly-Arg-Gly-AspSer-Pro [GRGDSP]). 6 -8 Exogenous fibronectin does not affect the proliferation of cultured corneal epithelial cells. 9 -13 The administration of fibronectin eye drops also facilitates corneal epithelial wound closure in experimental animals 14 -17 and has proven effective clinically for the treatment of persistent epithelial defects of the cornea. 5,18 -21 Fibronectin is a dimer of two almost identical polypeptides. It consists of several structural domains, including three types of repeat, that mediate the various biological functions of the protein. From the Departments of