The corneal stroma is an organized connective tissue with the major extracellular matrix component precisely arranged. There are at least three levels of corneal stromal architecture: (1) fibrils with a monotony of small diameters; (2) groups of well ordered fibrils with a constant spacing; and (3) orthogonal lamellae. The orderly development and maintenance of all three of these levels is essential for optical transparency. The interaction of different collagens is important in the control of fibril diameter immunoelectron microscopy and collagen type specific monoclonal antibodies will be used to study the distribution of type V collagen with type I collagen. Monoclonal antibodies against the terminal globular portion of type V collagen will be generated as probes in elucidating type V/I interactions in fibril structure. A self assembly assay will be used to study the interactions of types V and I collagen at various molar ratios in vitro. The organization of type VI collagen will be studied using immunoelectron microscopy and monoclonal antibodies. Type IV collagen may mediate cell-matrix or matrix-matrix interactions. Procollagen processing may be partially responsible for the control of collagen fibril diameter. The role of the amino and carboxy terminal propeptides of type I collagen and their processing in fibril assembly will be evaluated in situ using electron microscopy and antibodies against the propeptides and the specific processing enzymes. The influence of proteoglycans in the control of fibril diameter and the regulation of interfibrillar spacing will be studied in situ using ultrastructural histochemistry and biochemical techniques in normal corneas and after the in ovo inhibition of proteoglycan synthesis as well as in vitro using a self assembly assay and purified matrix components. The role of the corneal fibroblast in the development of matrix macro-architecture, specifically, the ability to reorganize matrix, intercalate newly formed fibrils and fibril bundles into an existing matrix and the addition of collagen types V and VI to an existing matrix will be studied using a collagen gel system. These studies on the mechanisms controlling corneal fibril structure and the generation of micro- and macro-architecture will contribute to our understanding of how the precise structure associated with corneal transpareny develops. This knowledge is fundamental to the possible manipulation of structure during growth, injury and repair.
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