Epithelial migration and/or adhesion to basement membrane and stroma appear to be compromised in persistent corneal epithelial defects. To better understand the processes of epithelial cell migration during the healing of corneal wounds, it is important to understand the types of adhesive interactions that exist between the epithelium and the underlying basement membrane or stroma during migration, and any changes in these interactions that occur as migration proceeds. A family of integral membrane, cell- surface glycoproteins called integrins (formerly termed """"""""fibronectin receptors"""""""", vitro-nectin receptors) function in mediating migration of cells during development and in adhesion of platelets and leukocytes to substrates. The extracellular domains of integrins bind to extracellular matrix components, and their cytoplasmic domains bind indirectly to the cytoskeleton.
Aim 1 of this proposal is to identify and quantitate the integrins expressed in stationary and migrating epithelia in an in vitro, rat organ culture corneal debridement model using subunit-specific integrin antisera (11 known integrin -chains, 4 known -chains). Integrins will be localized within corneal tissues by immunofluorescence or in situ hybridization. In addition, mRNA levels will be quantitated to determine if integrin expression is regulated by RNA accumulation or by translational mechanisms.
Aim 2 is to determine the effect of changes in the composition of the underlying substrate on the expression and localization of integrins. Corneas wounded by debridement or keratectomy will be evaluated in vitro and in vivo for integrins. To further determine the effect of substrate, epithelial migration rates and integrin expression will be correlated after sheets of intact epithelium are placed on purified matrix components.
Aim 3 is to evaluate the effect of inflammation, a known inhibitor of epithelial migration, on expression of integrins and their localization using two rat model systems of inflammation. One model will include addition of inflammatory cell extract of organ cultures of rat corneas after debridement or keratectomy. The second model will use a high-tyrosine diet to induce corneal inflammation in young rats. Inflamed corneas will be wounded and integrin production and epithelial migration assessed in organ culture. The proposed studies of integrin expression in corneal epithelium will provide insight into the basic cell biology of migration and the role of integrin:cell matrix interactions in the healing of corneal wounds.
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