To preserve the integrity of the corneal epithelium it is essential to maintain a transparent and refractile medium. Persistent epithelial defects and recurrent corneal erosions are painful and may occur if the integrity of the corneal surface is challenged by injury or disease. Epithelial wound healing becomes compromised when disorders in the attachment and synthesis of corneal epithelial cells are prevalent. The objectives of this proposal are to understand the mechanisms by which corneal epithelial cells form attachment sites and whether these sites are directly related to the presence of integral proteins that are synthesized when cells are seeded onto corneal substrates. Specifically the aims of the project are; 1) to examine the regulation of integrin synthesis ((an integral membrane protein) by epithelial cells in response to both normal and abnormal substrates in vitro using both biochemical (SDS-PAGE, fluorography, immunoblot and Western blot analysis and HPLC) and immunocytochemical techniques, 2) to examine the presence of integrin with respect to extracellular matrix and cytoskeletal proteins of rabbit and human cornea using immunoelectron microscopy and 3) to examine the response of epithelial cells to define substrate in vitro. In the last aim both the regulation of protein synthesis of integrin and cytoskeletal proteins and the MRNA levels of integrin on chemically defined substrate will be examined and correlated with morphology and cell growth. The second part of the last aim is to compare the levels of integrin mRNA expression when cells are seeded onto normal and abnormal corneal substrates using in situ hybridization. These will be compared to mRNA levels of integrin on normal and diseased human corneas. The adhesion assay develop by the applicant will be used as vehicle to examine the response of epithelial cells to their substrate morphologically and biochemically. By understanding how cells attach and regulate the synthesis of proteins in normal and abnormal corneas, one can potentially develop therapies for those with persistent epithelial defects and recurrent corneal erosions.
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