Purpose: Although overnight eye closure is known to result in hypoxia and release of potent angiogenic factors, even prolonged eye closure does not result in corneal neovascularization. This suggests that the closed eye tear film may contain factors that can impede neovascularization. Closed eye tear fluid is known to contain proteases capable of converting plasminogen/plasmin to angiostatin and other angiostatin-like A-chain fragments which are potent inhibitors of angiogenesis. This study was designed to characterize open and closed eye tear fluid for the presence of these entities.
Methods: Open and closed eye tears were collected by microcapillaries from normal individuals. Tears were centrifuged and the supernatants analyzed by SDS-PAGE and western blotting. Membranes were probed with antibodies specific for the A-chain of plasmin and plasminogen and with antibodies specific for conformational domains on the smaller N terminal kringles 1-->4 and kringles 1-->3 fragments which are known angiogenesis inhibitors. Supernatants were also analyzed after fractionation by HPLC and binding to lysine sepharose 4B. The isolated fragments were identified based on size, lysine-binding capabilities, antigenic properties and by comparison with standards.
Results: Open eye tear fluid from all normal individuals contained low levels of plasminogen, but no detectable antigens consistent with free A-chain or angiostatins. Tears collected after overnight eye closure contained significant amounts of plasminogen, A-chain antigen and various A-chain fragments including kringles 1-->4 and kringles 1-->3 and most likely free kringle 5, all known to have anti-angiogenesis properties. These were often present in concentrations likely to be physiologically significant. In samples collected from an atopic subject, the concentration of angiostatins in CTF increased markedly during active phases of the disease reaching levels of several ng/microl. In these instances and in similar samples obtained from other atopic individuals experiencing active reactions, angiostatin was often detectable in basal-type tear fluid.
Conclusion: A-chain fragments, which can inhibit angiogenesis, are often present at physiologically significant levels in human tear fluid collected after overnight eye closure. These fragments may play a role in preventing neovascularization in the hypoxic closed eye environment and may well be up regulated during inflammatory reactions.