Keratoconus (KC) is a disease characterized by corneal thinning and severe irregular astigmatism. In this country, it is a leading cause of visual morbidity and often requires corneal transplantation. The incidence of KC varies from 4 to 600 per 100,000 and the disease appears in all races. In the U.S., the incidence of KC is estimated to be 1 person per 2000. Typically onset occurs at an early age and can lead to a lifetime of disability. Mild KC is probably more prevalent in the general population than previously thought since more sophisticated methods of measuring corneal shape now allow earlier diagnosis. Recently, there is an additional interest in KC since some feel these patients are not suitable candidates for refractive surgeries which are being increasingly performed. While keratoconus may have multiple etiologies, our studies have demonstrated that approximately 75% are associated with an abnormal enzyme system which can degrade the corneal matrix. This system is composed of a constitutive corneal enzyme, matrix metalloproteinase-2 (MMP-2) and naturally occurring endogenous tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The first goal of this grant is to identify alterations in MMP-2 and/or TIMPs responsible for this elevated enzyme activity. The following will be determined: (a) primary structures of MMP-2 and TIMPs by cloning and sequencing, (b) secondary structures of MMP-2 and TIMPs by carbohydrate and electrophoretic analyses, and (c) the mechanism for lower TIMP-1 levels in KC by analyzing the translational efficiency and degradation rates. The second goal of the grant is to identify the substrate(s) within KC and normal corneas acted upon by MMP-2 which could lead to thinning. We will examine biochemically the degradation fragment profiles of the isolated components of corneas (collagens and proteoglycans) and intact corneas, after incubation with MMP-2. The third goal is to further characterize a recently described soluble factor(s) produced by KC cells which can depress MMP-2 transcript. This factor will be isolated by column chromatography and its properties further characterized. The effect this soluble factor has upon the transcription of MMP-2, TIMP-1 and TIMP-2 will be examined by Northern blot analyses. These results will significantly enhance the basic understanding of MMP-2 and its inhibitors, TIMP-1 and TIMP-2, in normal human corneas and also provide valuable information on the etiology of KC.