Corneal ulceration is a devastating disorder that destroys the corneal tissue and can cause blindness. Past research on this problem has yielded frustratingly little clinically applicable information. Our long-term goals are to identify and characterize cells and mediators that cause tissue destruction after corneal injury and infection, and to determine the molecular mechanisms regulating expression of these mediators. In preliminary experiments, four different metalloprotease species, synthesized and secreted by stromal fibroblasts, were identified:MMP1 (collagenase), MMP2 (type IV collagenase), MMP3 (stromelysin), and MMP4 (a new enzyme). Corneal epithelial cells also make MMP2 and MMP4; over- expression of these enzymes by corneal cells could cause stromal degradation. Since these enzymes can degrade substrates found in the epithelial basement membrane, their over-expression could lead to the types of epithelial defects that virtually always precede stromal degradation. Enzymatic activity in cornea might be further controlled, even after the metalloproteases are synthesized and secreted, via the specific regulatory molecule, tissue inhibitor of metalloproteinases (TIMP). TIMP could be delivered to the cornea via the circulatory system or could be synthesized and secreted by corneal fibroblasts. Preliminary experiments and results in the literature show that the metalloprotease/TIMP are each individually regulated in response to different stimuli. Thus, studies designed to understand the role of these molecules in corneal stromal degradation must consider metalloprotease/TIMP expression as a group, rather than individually. In the proposed studies, a set of antibody and cDNA probes for the metalloproteases and TIMP will be completed by raising antiserum to MMP4, developing a cDNA probe for MMP4, and raising antiserum to TIMP. These probes will then be used for in situ hybridization and immunolocalization studies on rabbit and human corneal tissue. The level, location, and timing of expression of metalloproteases/TIMP by corneal cells will be determined during the normal events of corneal wound-healing, and the degree that the circulation contributes TIMP to the cornea will also be examined. This appropriate expression and localization will be compared with the situation during the process of corneal ulceration to understand how abnormal control of metalloprotease expression by corneal cells might lead to this disorder. Finally, experiments will be directed toward understanding how synthesis of these metalloproteases/TIMP might be controlled on the molecular level. These final studies will particularly focus on the enzymes about which least is currently known, MMP2 and MMP4.
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