Fundamental to the understanding of corneal function and its pathobiology is the elucidation of the macromolecular and cell-matrix interactions involved in the development and maintenance of the corneal stroma. The long term objectives of this proposal are to characterize the mechanisms involved in the assembly of tissue-specific extracellular matrices during corneal development. The general hypotheses are: (1) that the stoichiometry and interactions of collagen types I/V during fibrillogenesis are the major mechanisms regulating corneal fibril diameter and may also mediate matrix-matrix or cell-matrix interactions; (2) that interactions of decorin and lumican with other matrix components are important in the regulation of the later stages of matrix assembly; (3) type VI collagen mediates cell-matrix and matrix-matrix interactions that are important in the stabilization and integration of the stroma; and (4) cell-matrix interactions define the pathway presumptive corneal fibroblasts follow, provide cues for differentiation and direct tissue-specific matrix assembly during corneal development. These hypotheses will be tested by altering the matrix components both in vivo and in vitro using recombinant retroviral constructs to express dominant-negative mutations or antisense RNA. These will be correlated with biochemical, molecular, immunochemical and morphological analyses. Fibril and matrix structure will be analyzed using ultrastructural and immunochemical methods. Matrix interactions will be investigated using molecular binding assays as well as cell attachment and migration assays. The differentiation of corneal fibroblast will be studied using culture models and followed using type V collagen, lumican and a developmentally regulated antigen as markers. The understanding of corneal development transparency as well as disorders such as would healing and some dystrophies requires a knowledge of the mechanisms involved in the control of collagen fibrillogenesis, matrix assembly and development of tissue-specific architecture.
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