PB1342 - Anti-angiogenic activity of novel plasminogen fragments produced by a bacterial metalloproteinase

Background: Tumor growth depends on the development of angiogenesis, a process of new vessel formation from pre-existing normal blood vessels. Tumor-derived angiogenesis occur through the release of growth factors from the tumor. Proteolytic fragments of plasminogen, containing various number of kringle domains of plasminogen, collectively called angiostatin, are a naturally occurring inhibitor of angiogenesis and inhibits tumor growth. Different forms of angiostatin have different activities. In our previous studies, we found a novel bacterial protease (bacillolysin MA; BLMA) that specifically cleaves plasminogen at the Ser441−Val442 bond, yielding an angiostatin species comprising of Glu1−Ser441 polypeptide (BL-angiostatin).

Aims: The objective of this study was to characterize the anti-angiogenic activity of BL-angiostatin and seek for a method of the use of BL-angiostatin/BLMA in tumor treatment.

Methods: BL-angiostatin was prepared by BLMA cleavage of human plasminogen. Angiogenesis was assayed by tube formation and proliferation of vascular endothelial cells in vitro and by the mouse dorsal air sac model in vivo. Tumor growth was assessed by subcutaneously implanting Lewis lung carcinoma cells in mice.

Results: BL-angiostatin inhibited in vitro angiogenesis at 1 g/mL and suppressed in vivo tumor growth at 0.3 to 10 mg/kg. These activities were considerably higher than those of recombinant angiostatin (kringles 1−3). Unlike recombinant angiostatin, BL-angiostatin has the N-terminal PAN domain and one or two sugar chains with a terminal sialic acid. The removal of the PAN domain or the terminal sialic acid decreased anti-angiogenic activity in vivo, suggesting roles for the PAN and terminal sialic acids in the anti-angiogenic activity. A direct intravenous injection of BLMA at 2 mg/kg produced angiostatin in vivo in mice and suppressed an early-stage tumor growth.

Conclusion(s): These results raise a possibility for the use of BLMA in the treatment of tumor through in vivo generation of angiostatins.