Abstract
BackgroundAngiostatin, an endogenous angiogenesis inhibitor, is a fragment of plasminogen. Its anti-angiogenic activity was discovered with functional assays in vivo, however, its direct action on endothelial cells is moderate and identification of definitive mechanisms of action has been elusive to date. We had previously demonstrated that innate immune cells are key targets of angiostatin, however the pathway involved in this immune-related angiogenesis inhibition was not known. Here we present evidence that IL-12, a principal TH1 cytokine with potent anti-angiogenic activity, is the mediator of angiostatin's activity.MethodsFunction blocking antibodies and gene-targeted animals were employed or in vivo studies using the subcutaneous matrigel model of angiogenesis. Quantitative real-time PCR were used to assess modulation of cytokine production in vitro.ResultsAngiostatin inhibts angiogenesis induced by VEGF-TNFα or supernatants of Kaposi's Sarcoma cells (a highly angiogenic and inflammation-associated tumor). We found that function-blocking antibodies to IL-12 reverted angiostatin induced angiogenesis inhibition. The use of KO animal models revealed that angiostatin is unable to exert angiogenesis inhibition in mice with gene-targeted deletions of either the IL-12 specific receptor subunit IL-12Rβ2 or the IL-12 p40 subunit. Angiostatin induces IL-12 mRNA synthesis by human macrophages in vitro, suggesting that these innate immunity cells produce IL-12 upon angiostatin stimulation and could be a major cellular mediator.ConclusionOur data demonstrate that an endogenous angiogenesis inhibitor such as angiostatin act on innate immune cells as key targets in inflammatory angiogenesis. Angiostatin proves to be anti-angiogenic as an immune modulator rather than a direct anti-vascular agent. This article is dedicated to the memory of Prof Judah Folkman for his leadership and for encouragement of these studies.
Highlights
Angiostatin, an endogenous angiogenesis inhibitor, is a fragment of plasminogen
Angiomotin appears to be involved in VEGF signaling in vitro and angiomotin deletion is associated with variable degrees of vascular malformation in vivo [14] AST seems to have no effect in the same system [15]
Effects of function blocking antibodies on angiogenesis in vivo In a preliminary study we noted elevation of serum IL-12 in tumor-bearing animals treated locally with AST (Fig. 1b), suggesting that this potent anti-angiogenic cytokine may play a role in the effects of AST
Summary
Angiostatin, an endogenous angiogenesis inhibitor, is a fragment of plasminogen. Its antiangiogenic activity was discovered with functional assays in vivo, its direct action on endothelial cells is moderate and identification of definitive mechanisms of action has been elusive to date. Angiostatin and related forms consisting of the first 1–5 kingles in plasminogen (here termed collectively AST) is generated by the action of diverse proteases, including metalloproteases (MMP2, MMP12, MMP9) and serine proteases (PSA, neutrophil elastase) [3,4]. These enzymes are subject to precise regulation, and are typically activated during tumor invasion, angiogenesis and inflammation, AST is produced only under certain conditions and it could represent an important modulator of homeostatic responses. Following identification with in vivo studies, numerous in vitro studies have sought to identify the effects of AST on endothelial cells. Angiomotin appears to be involved in VEGF signaling in vitro and angiomotin deletion is associated with variable degrees of vascular malformation in vivo [14] AST seems to have no effect in the same system [15]
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