An Inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface.

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.

Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for k1-5-induced antiangiogenesis.

We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the alpha- and beta-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.

Angiostatin and anti-angiogenic therapy in human disease.

Many diseases have abnormal quality and/or quantity of vascularization as a characteristic feature. Cancer cells elicit the growth of new capillaries during neovascularization in a process termed angiogenesis. In diabetics, pathologic angiogenesis in various tissues is a clinical feature of many common complications. Therefore, the diabetic cancer patient warrants special consideration and extra care in the design of anti-angiogenic treatments without adverse side effects. Some treatment regimens that look promising in vitro, in animal models, or in early clinical trials may be contra-indicated for diabetics. This chapter will review the common complications of diabetes, with emphasis on the angiogenic pathology. Recent research related to the mechanism of action and basis for specificity of the anti-angiogenic peptide, angiostatin, will be the focus. The aim is to shed light on areas in which more research is needed with respect to angiostatin and other anti-angiogenic agents and the microenvironmental conditions that affect their activities, in order to develop improved therapeutic strategies for diabetic cancer patients.

The mechanism of action of angiostatin: can you teach an old dog new tricks?

What is angiostatin? In 1994, Folkman and colleagues published a landmark paper describing anti-tumor effects in mice with a purified fragment of plasminogen they named angiostatin (1). Although many papers have been published describing activities of cryptic polypeptides derived from plasminogen fragments, this was the first report which associated plasminogen kringles 1-4 as a suppressor of metastasis development. This review will describe what is known about the mechanism of action of angiostatin from the current literature.