Elevated levels of urine angiostatin and plasminogen/plasmin in cancer patients.

Previously, a specific angiogenesis inhibitor, angiostatin, discovered in urine and serum of tumor-bearing mice, was reported to potently block tumor growth and metastasis in animal models. Detection of angiostatin and its precursor proteins in urine from cancer patients has not been reported. Now, we report the development of an antibody-based analysis system that allows us to detect angiostatin and plasminogen/plasmin (Pgn/plasmin) in the urine of cancer patients. The detection system is a combination of a novel lysine-ELISA assay and Western immunoblot analysis using a specific antibody to human angiostatin and Pgn/plasmin. High levels of Pgn/plasmin were detected in the urine from various cancer patients, whereas healthy individuals showed relatively low levels of urine Pgn/plasmin. Of interest, angiostatin is detectable in urine samples of patients with various cancers, including acute lymphoblastic leukemia, suggesting that angiogenesis may play an important role in the development and progression of leukemia. Our data for the first time show that angiostatin and Pgn/plasmin are present at relatively high levels in the urine of human cancer patients. Detection of urine angiostatin in cancer patients helps us not only to understand the role of this angiogenesis inhibitor in cancer development and progression but also allows us to develop tools of cancer diagnosis and prognosis. Thus angiostatin has both therapeutic and diagnostic implications in cancer disease.

Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis.

Proteolytic enzymes are involved in generation of a number of endogenous angiogenesis inhibitors. Previously, we reported that angiostatin, a potent angiogenesis inhibitor, is a proteolytic fragment containing the first four kringle modules of plasminogen. In this report, we demonstrate that urokinase-activated plasmin can process plasminogen to release an angiogenesis inhibitor, K1-5 (protease-activated kringles 1-5). K1-5 inhibits endothelial-cell proliferation with a half-maximal concentration of approximately 50 pM. This inhibitory effect is endothelial-cell-specific and appears to be at least approximately 50-fold greater than that of angiostatin. A synergistic efficacy of endothelial inhibition was observed when angiostatin and kringle 5 (K5) were coincubated with capillary endothelial cells. The synergistic effect is comparable to that produced by K1-5 alone. Systemic treatment of mice with K1-5 at a low dose significantly blocked the fibroblast growth factor-induced corneal neovascularization, whereas angiostatin had no effect at the same dose. K1-5 also suppressed angiogenesis in chicken embryos. Systemic administration of K1-5 at a low dose at which angiostatin was ineffective significantly suppressed the growth of a murine T241 fibrosarcoma in mice. The antitumor effect correlates with the reduced neovascularization. These findings suggest that the plasmin-mediated proteolysis may be involved in the negative switch of angiogenesis.