Macrophage formation of angiostatin during inflammation. A byproduct of the activation of plasminogen.
Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin directly or in cooperation with tumor-associated macrophages. In studies reported here, we determined whether angiostatin is generated in mice under non-neoplastic settings. Utilizing murine RAW264.7 macrophages and thioglycollate-elicited peritoneal macrophages, we demonstrate that angiostatin-like fragments are generated as a byproduct of the proteolytic regulation of membrane-bound plasmin. Plasmin proteolysis and subsequent loss in membrane-bound plasmin activity requires active plasmin but was unaffected by inhibitors of metalloproteinases. Lysine binding fragments of plasmin, isolated from macrophage-conditioned media utilizing affinity chromatography, appeared as a major (48 kDa) and two minor bands (42 and 50 kDa) in SDS-polyacrylamide gel electrophoresis and were immunoreactive with anti-kringle 1-3 IgG. Each peptide begins with Lys77 and contains the entire sequence of angiostatin. The affinity isolated plasmin fragments inhibited bFGF-induced endothelial cell proliferation. Lavage fluid recovered from the peritoneal cavities of mice previously injected with thioglycollate contained angiostatin-like plasmin fragments similar to those generated in vitro. This is the first demonstration that angiostatin-like plasmin fragments are generated in a non-neoplastic inflammatory setting. Thus, in addition to regulating pericellular plasmin activity, proteolysis of plasmin generates inactive kringle-containing fragments expressing angiostatic properties.
Falcone
D J
DJ
Department of Pathology, Cornell Medical College, New York, New York 10021, USA. dfalcone@mail.med.cornell.edu
Khan
K M
KM
Layne
T
T
Fernandes
L
L
D043168:Angiostatins; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects; D004789:Enzyme Activation; D005122:Exudates and Transudates; D005341:Fibrinolysin / Q000378:metabolism*; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism*; D018082:Kringles; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism* / Q000494:pharmacology; D013864:Thioglycolates / Q000494:pharmacology
D008565:Membrane Proteins; D010446:Peptide Fragments; D013864:Thioglycolates; D043168:Angiostatins; D010958:Plasminogen; D005341:Fibrinolysin; D008666:Metalloendopeptidases