KALLISTATIN IN HUMAN OCULAR-TISSUES - REDUCED LEVELS IN VITREOUS FLUIDS FROM PATIENTS WITH DIABETIC-RETINOPATHY

Purpose. Kallistatin is a serine proteinase inhibitor, which binds to tissue kallikrein and inhibits its proteolytic activity. This study is to determine the expression, cellular localization and the potential function of kallistatin in the eye. Methods. Tissue kallikrein-kallist atin complex formation was performed to detect the kallikrein-binding activity in ocular tissues. Immunoreactive kallistatin was detected an d quantified by an enzyme-linked immunosorbent assay using polyclonal antibody specific to human kallistatin. In situ hybridization histoche mistry was employed to localize the kallistatin mRNA in human eyes usi ng an antisense riboprobe of kallistatin. Results. We have identified active kallistatin in the cornea, ciliary body, sclera, choroid, optic nerve, retina, vitreous and aqueous fluids. Kallistatin binds to tiss ue kallikrein and forms an SDS-stable complex. Immunoreactive kallista tin was identified in these tissues. Linear dose-dependent curves of t he tissue extracts of the retina and choroid are parallel to that of p urified human kallistatin, suggesting their immunological identity. Th e kallistatin mRNA was identified in the ciliary muscle, lens epitheli al cells, all the layers of retina cells, optic nerve, choroid and vas cular endothelial cells. These cells were not stained by the sense rib oprobe under the same conditions, indicating the specificity of the hy bridization. We also compared immunoreactive kallistatin levels in vit reous fluids from 18 patients with diabetic retinopathy and 17 non-dia betic subjects. The results show that diabetic subjects have significa ntly lower kallistatin levels (233.0 +/- 14.6 ng/mg protein) compared to non-diabetic subjects (334.1 +/- 26.9 ng/mg protein). Conclusions. Kallistatin is produced endogenously in the eye and the decrease in th e vitreous kallistatin levels may be involved in diabetic retinopathy.