The long-range goal of the grant is to understand the role of corneal keratocyte crystallin proteins in modulation of corneal transparency at the cellular level. Recent studies have shown that rabbit corneal keratocytes abundantly express two water soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1 (ALDH1) in vivo. Furthermore, in preliminary studies of corneal wound healing and neonatal development in the rabbit, expression of these two proteins is highly correlated inversely to the backscattering of light from keratocytes in the intact, living cornea. Based on these data the PI proposes that TKT and ALDH1 in the rabbit keratocyte belong to a new class of crystalline proteins unique to the keratocyte, which serve a similar structural function in modulating corneal transparency, as do related crystalline proteins in the lens. Overall, the PI proposes that expression of keratocyte crystallin proteins may control corneal transparency at a cellular level, and therefore, play a role in the development of corneal clouding and haze following disease and injury, particularly after excimer laser photorefractive keratectomy, PRK. The PI will test the hypothesis through the following experimental aims: (1) identify and characterize putative keratocyte crystalline proteins from human, bovine, porcine, murine and avian corneas by SDS-Page, tryptic peptide sequence analysis and western blotting to verify the taxon specific expression of keratocyte crystalline proteins; (2) establish the developmental expression of keratocyte crystalline proteins and determine the relative contribution of crystalline protein expression to the neonatal development of corneal transparency following eyelid opening compared to tissue hydration, keratocyte cell density, size, thickness, and differentiation, and collagen fibril diameter and spacing; (3) determine the temporal expression of keratocyte crystalline proteins during repair of corneal freeze injury and correlate the expression of keratocyte crystallins to backscattering of light from keratocytes in intact, living cornea; (4) establish expression of keratocyte crystallins in vitro and determine the effects of light/dark and cytokine stimulation (TGFbeta, IL1, FGF2, PDGF) on crystalline protein expression; and (5) modulate keratocyte crystalline protein expression in vivo by topical application of cytokines or anti-cytokine blocking antibodies shown to upregulate or block crystallin expression and determine the effect on light scattering and corneal haze following PRK.
