TYPE IV COLLAGEN PEPTIDES--RECEPTORS IN CORNEAL FUNCTION

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The cornea is an attractive wound healing model because it has relatively simple and unique wound healing characteristics. The proposed studies will use type IV collagen purified fragments and chemically synthesized peptides with an objective of defining the molecular mechanisms involved in corneal epithelial cell adhesion and cell migration. The ultimate goal of these studies will be to develop peptide pharmaceuticals to modulate corneal wound healing. The information learned from these studies may have application to wound healing in general. In these studies we will evaluate the adhesion and spreading of corneal epithelial cells to type IV collagen, defined type IV collagen proteolytic fragments and synthetic peptides derived from type IV collagen. Next, we will evaluate the ability of chemically synthesized peptides from different regions of type IV collagen to stimulate the migration of corneal epithelial cells. In these studies both haptotaxis (migration to substratum attached) and chemotaxis (migration to solution phase ligands) will be determined. Integrins are a major class of cell surface receptors for extracellular matrix and basement membrane molecules. Integrins are complex heterodimers with important biological functions. We will define the integrin "receptors" on human corneal epithelial cells that mediate adhesion and migration to type IV collagen and specific synthetic peptide ligands derived from type IV collagen. a battery of specific anti- alpha chain antibodies and specific anti-beta chain antibodies will be used to delineate the full integrin complement. Lastly, we will test the influence of highly purified type IV collagen domains or fragments and chemically synthesized peptides derived from type IV collagen on the migration of rabbit corneal epithelial cells in the in vitro organ culture assay system. We have found three distinct synthetic peptides derived from the type IV collagen amino acid sequence that promote corneal cell adhesion: HEP-1 and HEP-2 from the NC1 region, and HEP-3 from the triple helical region. The HEP- 3 peptide, which promotes corneal epithelial cell adhesion, appears to represent a sequence of type IV collagen that is very important for stimulating cell migration. It seems likely that other sequences exist within the helical fragment in areas not yet tested that may also have cell adhesion or cell migration promoting capacity. These findings contribute to the molecular dissection of basement membrane structure and function, and they may serve as the basis for developing therapeutic agents to modify corneal epithelial behavior so as to have a salutary effect in problematic clinical conditions.