Immunohistochemical Localization of Counterreceptors for Galectins–3 and –7 in Normal and Healing Corneas

Abstract

Abstract: : Purpose: We have recently demonstrated that two carbohydrate–binding proteins, galectins–3 and –7, are among the key molecules that mediate corneal epithelial cell migration and re–epithelialization of corneal wounds (J. Biol. Chem. 277: 42299–42305, 2002). Galectins constitute a family of widely distributed carbohydrate–binding proteins known for their role in cell–matrix interactions, cell proliferation, and apoptosis. They are characterized by their affinity for ß–galactoside containing glycans found on many cell surface and extracellular matrix glycoproteins. Such glycoproteins located on the corneal surface and cell matrix may serve as counterreceptors for galectins. The interactions between galectins and counterreceptors are likely to play a key role in corneal epithelial cell migration during wound healing. In an effort to understand the molecular mechanism by which galectins–3 and –7 mediate corneal epithelial cell migration, the aim of the present study was to localize their counterreceptors in normal and healing corneas. Methods: Transepithelial excimer laser corneal ablations were produced on the right eye of each anesthetized mouse. Wounds were allowed to partially heal in vivo for 16–18 h. The left eye served as normal control. Eyes were excised out, embedded in OCT compound, and processed for preparation of frozen sections. For immunohistochemical analysis, tissue sections were incubated with biotinylated galectins–3 and –7. Staining was elicited using a freshly prepared avidin D and biotin–peroxidase complex and a DAB substrate. Negative control sections were incubated with galectins in the presence of a competing sugar, ß–lactose. Results: In both normal and healing corneas, galectins–3 and –7 stained corneal epithelium, epithelial basement membrane, stromal matrix, and keratocytes (n=4 corneas each, normal & healing). In each case, staining was abolished by a competing sugar, ß–lactose. Conclusions: In contrast to galectins–3 and –7, which are expressed only in corneal epithelium, counterreceptors of both galectins are expressed not only on corneal epithelial cells, but also on keratocytes. These data lead us to propose that galectins released from the epithelium during injury have the potential to modulate the function of keratocytes in a paracrine fashion. Future studies will attempt to further identify and characterize these counterreceptors in an effort to understand the mechanisms by which galectins influence corneal wound healing.

Keywords: cell adhesions/cell junctions • glycoconjugates/glycoproteins • wound healing