The extracellular matrix in corneal stroma is a highly ordered structure believed to play a major role in maintaining the transparency and rigidity of the cornea. How the components of the extracellular matrix are assembled and what molecular and cellular control mechanisms are involved in the formation of the matrix are only beginning to be understood.
The aim of this proposal is to study how heterotypic fibrils of collagen are assembled in human cornea. Normal human keratocyte cultures would be used as a model of collagen are assembled in human cornea and as a model system to study heterotypic fibril formation. Mutant keratoconus keratocyte cultures deficient in types I, III and V collagen synthesis would also be used in these studies. The project would use immunofluorescence microscopy, high voltage immunoelectron microscopy (HVEM), biochemical and molecular biology techniques to study the formation of heterotypic fibrils. The structure and formation of heterotypic fibrils of types I and III collagen and types I and V collagen would be studied using antibodies specific for types I, III and V collagen. Antibodies specific for fibronectin and types II and VI collagen would be used to see if these proteins interact with developing heterotypic fibrils. The incorporation of fibronectin and types I, II, III, V and VI collagen into matrix of subconfluent keratocyte cultures would be followed over a 96 hour period. Immunofluorescence microscopy and HVEM would be used to identify the composition of matrix fibrils and to determine the banding pattern and diameter of collagen fibrils in these cultures. RNA and Elisa analysis would be used to quantitate the amount of types I, III and V collagen incorporation into fibrils. In situ ultrastructural studies on normal and keratoconus corneal buttons would also be done to correlate in vivo collagen binding patterns, fibril diameter and composition of heterotypic fibrils to those observed in culture. Finally, to study the function of types III of V collagen in the formation of heterotypic fibrils, antisense mRNAs for types III and V collagen would be developed and inserted into keratocytes to selectively turn on, or off, type III or type V collagen synthesis.
