Binding of Topically Applied Human Annexin A5 on Rabbit Corneal Epithelial Wound

Abstract

Abstract: : Purpose:Human annexin A5, a calcium-dependent phospholipid binding protein, promotes the migration of corneal epithelial cells in vitro and corneal wound healing on de-epithelialized rabbit cornea in vivo. The purpose of this study is to compare the pharmacokinetics of topically instilled annexin A5 on intact and de-epithelialized rabbit eyes, and to determinine whether human annexin A5 binds to the basement membrane of wounded cornea. Methods:In pharmacokinetic study,¹²⁵I-labeled recombinant human annexin A5 ( 300µg/mL, 50µL/time ) was instilled on intact and de-epithelialized rabbit eyes four times at 1 hour interval. After the final instillation, ocular distribution of radioactivity was evaluated by whole-head autoradiography. In annexin A5 binding study, the binding of biotinylated human annexin A5 to the components of the corneal basement membrane (type IV collagen, type VII collagen and laminin) was detected by enzyme-linked immunosorbent assay (ELISA). Results: In the instillation of ¹²⁵I- human annexin A5 on intact eyes, the whole-head autoradiographphy showed that the radioactivity at 1 hour was observed on cornea and conjunctiva, but no radioactivity was observed at 24 hours. In de-epithelialized eyes, the radioactivity was detected at the wounded area of cornea at 24 hours after the final instillation. At 72 hours, the wounded area was covered by the epithelium, but the radioactivity was still detected on the cornea. The half-life of the radioactivity in the intact cornea after the repeated instillations was 0.5 days, while that of the de-epithelialized cornea was 4.6 days. There was no significant difference in the blood parameters of annexin A5 between intact and de-epithelized rabbits. Human annexin A5 binds to type IV collagen, type VII collagen and laminin. Conclusions:Topically instilled annexin A5 in de-epithelial cornea may bind to the basement membrane.

Keywords: wound healing • cornea: epithelium • animal model