Diurnal variations in angiostatin in human tear fluid: A possible role in prevention of corneal neovascularization

Purpose. Although overnight eye closure is known to result in hypoxia and r elease of potent angiogenic factors, even prolonged eye closure does not re sult in corneal neovascularization. This suggests that the closed eye tear film may contain factors that can impede neovascularization. Closed eye tea r fluid is known to contain proteases capable of converting plasminogen/pla smin 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 speci fic for the A-chain of plasmin and plasminogen and with antibodies specific for conformational domains on the smaller N terminal kringles 1-->4 and kr ingles 1-->3 fragments which are known angiogenesis inhibitors. Supernatant s were also analyzed after fractionation by HPLC and binding to lysine seph arose 4B. The isolated fragments were identified based on size, lysine-bind ing capabilities, antigenic properties and by comparison with standards. Results. Open eye tear fluid from all normal individuals contained low leve ls of plasminogen, but no detectable antigens consistent with free A-chain or angiostatins. Tears collected after overnight eye closure contained sign ificant amounts of plasminogen, A-chain antigen and various A-chain fragmen ts including kringles 1 -->4 and kringles 1-->3 and most likely free kringl e 5, all known to have anti-angiogenesis properties. These were often prese nt in concentrations likely to be physiologically significant. In samples c ollected from an atopic subject, the concentration of angiostatins in CTF i ncreased markedly during active phases of the disease reaching levels of se veral ng/mu l. In these instances and in similar samples obtained from othe r atopic individuals experiencing active reactions, angiostatin was often d etectable in basal-type tear fluid. Conclusion. A-chain fragments, which can inhibit angiogenesis, are often pr esent at physiologically significant levels in human tear fluid collected a fter overnight eye closure. These fragments may play a role in preventing n eovascularization in the hypoxic closed eye environment and may well be up regulated during inflammatory reactions.