| Corneal opacity leaves 1.5 million people visually impaired worldwide. One potential key player in the formation and maintenance of corneal transparency is a keratan sulfate proteoglycan lumican. The role of lumican in the formation and maintenance of bio-glass, transparent cornea, was examined using three different experimental systems. First, a wound healing system was used to understand the expression of lumican by corneal cells during stromal matrix restructuring and remodeling. Three different types of wounds were generated on mouse corneas: partial epithelial debridement, total epithelial debridement, and alkali burn wounds. Lumican expression was analyzed over a 12 week time course. Second, cultured corneal fibroblasts were used in an ex vivo expression system to determine the impact of lumican on collagen fibrillogenesis. Cultured corneal fibroblasts were stably transfected with either a wildtype lumican (lumWT) or 41cysteine to serine mutated lumican (lumC/S). Under proper conditions stable transformants formed a three-dimensional extracellular matrix over a 4 or 6 week time period. Finally, DNA microinjection into the corneal stroma was used as an in vivo expression system. Our results demonstrate the following: (1) the amount of lumican expression by corneal stroma cells in a wound healing situation decreases during stromal remodeling and is also dependent on the severity of the wound. This finding indicates that the gene expression pattern of keratocytes is lost during the wound healing response. (2) Mutation of 41Cys to serine is adequate to impair the role of lumican in ex vivo collagen fibrillogenesis. The actual interaction of lumican with collagen is disrupted by this single amino acid mutation. The actual impact is possibly due to a structural change caused by this mutation. (3) Plasmid DNA microinjection into the corneal stroma is an effective tool to drive protein expression and is adequate to rescue the thin corneal stroma phenotype of lumican null mice. DNA microinjection into the corneal stroma is capable of being an efficient and safe way to serve as a gene therapy strategy in treating ocular surface diseases. This technique is simple, non-invasive, and effective in expressing a therapeutic protein of interest. |
