Antiangiogenic proteins require plasma fibronectin or vitronectin for in vivo activity

Abstract

Fragmentation of various extracellular matrix and blood proteins generates antiangiogenic substances that are physiological regulators of angiogenesis. Some of these compounds are in clinical trials as inhibitors of tumor angiogenesis. Anastellin, an antiangiogenic protein fragment derived from fibronectin, was unable to inhibit matrigel plug angiogenesis in mice that lack plasma fibronectin. Anastellin was fully active in mice that are null for vitronectin, which, like fibronectin, is a major adhesion protein in the blood. An antiangiogenic form of antithrombin showed the opposite pattern. The activity of endostatin was impaired in both fibronectin-and vitronectin-deficient mice. These results suggest a shared mechanism of action for antiangiogenic factors derived from extracellular matrix and plasma proteins: these factors form complexes with adhesion proteins in plasma to create an active antiangiogenic substance.A growing class of antiangiogenic substances is derived from extracellular matrix and blood proteins by proteolysis or other modifications. These substances include fragments from thrombospondin (1), plasminogen (angiostatin; ref. (5), and the fibronectin fragment anastellin (6, 7). The molecular mechanisms whereby these substances exert their antiangiogenic activities are poorly understood. Anastellin binds to and polymerizes fibronectin and fibrinogen (8, 9). The antiangiogenic form of antithrombin is similar to the modified antithrombin that binds vitronectin (10, 11). Vitronectin, like fibronectin, is an arginine-glycine-aspartic acid (RGD)-containing adhesion protein present in plasma (12). Other angiogenesis inhibitors also interact with one or more adhesion proteins: angiostatin and its parent protein, plasminogen, bind vitronectin (13), whereas endostatin binds fibulins and nidogen-2 (14).The interactions of the various angiogenesis inhibitors with adhesion proteins led us to hypothesize that adhesion protein binding could underlie antiangiogenic activity, and that activities of the various inhibitors could converge on this ability to form such adhesion protein complexes (7). To test this hypothesis, we examined the two antiangiogenic proteins with the most extensively documented adhesion protein interactions, anastellin and antithrombin. We used angiogenesis induced by implanting matrigel (basement membrane) plugs (15, 16) into mutant mice that conditionally lack plasma fibronectin (17) or have their vitronectin gene knocked out (18).We show here that anastellin does not suppress angiogenesis in matrigel plugs implanted into mice that lack plasma fibronectin. In contrast, the antiangiogenic form of antithrombin is inactive in mice that lack vitronectin. Endostatin is essentially inactive in mice that lack either plasma fibronectin or vitronectin. These results strongly support the hypothesis that the activity of extracellular matrix-and blood protein-derived angiogenesis inhibitors depends on interactions with adhesion proteins. Materials and MethodsProteins. Anastellin (a fragment from the...

Antiangiogenic treatment with endostatin inhibits progression of AML in vivo

“…Specific RGD motifs have been identified as potential binding partners for ES and similar antiangiogenic proteins. 35 Experiments performed in order to test effects of ES delivered by Alzet pumps on endothelial permeability in vivo showed inhibition of VEGF-induced permeability only after 3 days of treatment (unpublished data), supporting that ES has to saturate all binding targets before a systemic effect can be observed. Since systemic concentration of ES reached by microbeads is only slightly elevated over normal levels, we speculate that selective accumulation in sensitive endothelia achieved by continuous delivery might be sufficient for the effect observed.…”

Endogenous Inhibitors of Angiogenesis

“…Interestingly, the activity of endostatin is impaired in both fibronectindeficient and vitronectin-deficient mice. This suggests a shared mechanism of action for antiangiogenic factors derived from ECM and plasma proteins (66).…”

Domain Unfolding Plays a Role in Superfibronectin Formation

“…Subsequent studies of anastellin have revealed potentially important biological activities when injected peritoneally into mice, including inhibition of angiogenesis and tumor growth (17). These activities required plasma FN (18), so it is likely that sFN plays a role. When tested in cell culture, Bourdoulous et al (19) found that high concentrations of anastellin (20 M) blocked FN matrix formation and caused an established matrix to disappear after 16 h of treatment.…”