Angiostatin, an internal fragment of plasminogen, is a potent inhibitor of angiogenesis, which selectively inhibits endothelial cell proliferation. When given systemically, angiostatin potently inhibits tumor growth and can maintain metastatic and primary tumors in a dormant state defined by a balance of proliferation and apoptosis of the tumor cells. We identified angiostatin while studying the phenomenon of inhibition of tumor growth by tumor mass and have elucidated one mechanism for this phenomenon. In our animal model, a primary tumor almost completely suppresses the growth of its remote metastases. However, after tumor removal, the previously dormant metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kD plasminogen fragment which we have sequenced and named angiostatin. Human angiostatin, obtained from a limited proteolytic digest of human plasminogen, has similar activities. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases and primary tumors. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by the angiogenesis inhibitor angiostatin.

Angiostatin, a 38 kDa internal fragment of plasminogen, is an antiangiogenic endothelial cell inhibitor. It regresses several primary and metastatic tumors in mice. To produce recombinant angiostatin for further structural and functional studies, the mouse angiostatin gene preceded by a sequence including a signal peptide of plasminogen was introduced into baculovirus. Recombinant murine angiostatin was purified from the culture medium of angiostatin baculovirus-infected insect cells (yield = 1 mg/liter) with a single-step of lysine-Sepharose chromatography. The angiostatin baculovirus-infected insect cells expressed and secreted a 52 kDa polypeptide that demonstrated all of the biological activities of angiostatin. A partial amino acid sequence of the NH2-terminus of the secreted protein revealed that the signal peptide was recognized and properly cleaved in insect cells. The recombinant murine angiostatin potently inhibited the proliferation of bovine capillary endothelial cells in vitro (half maximal inhibition = 50 ng/ml) and suppressed the growth of primary Lewis lung carcinoma in vivo (6 mg/kg/day, T/C = 0.08).

Recently we have identified angiostatin, an endogenous angiogenesis inhibitor of 38 kDa which specifically blocks the growth of endothelial cells (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; Folkman, J. (1995) Nat. Med. 1, 27-31). Angiostatin was shown to represent an internal fragment of plasminogen containing the first four kringle structures. We now report on the inhibitory effects of individual or combined kringle structures of angiostatin on capillary endothelial cell proliferation. Recombinant kringle 1 and kringle 3 exhibit potent inhibitory activity with half-maximal concentrations (ED(50)) of 320 nM and 460 nM, respectively. Also, recombinant kringle 2 displays a significant inhibition, although decreased compared with both kringle 1 and kringle 3. In contrast, kringle 4 is an ineffective inhibitor of basic fibroblast growth factor-stimulated endothelial cell proliferation. Among the tandem kringle arrays, the recombinant kringle 2-3 fragment exerts inhibitory activity similar to kringle 2 alone. However, relative to kringle 2-3, a marked enhancement in inhibition is observed when individual kringle 2 and kringle 3 are added together to endothelial cells. This implies that it is necessary to open the cystine bridge between kringle 2 and kringle 3 to obtain the maximal inhibitory effect of kringle 2-3. An increased (<2-fold) inhibitory activity is observed for the kringle 1-3 fragment (ED(50) = 70 nM) compared with kringle 1-4 (ED(50) = 135 nn). These data indicate that the anti-proliferative activity of angiostatin on endothelial cells is shared by kringle 1, kringle 2, and kringle 3, but probably not by kringle 4 and that more potent inhibition results when kringle 4 is removed from angiostatin. Thus, in view of the variable lysine affinity of the homologous domains, it would appear that lysine binding capability does not correlate with the relative inhibitory effects of the kringle-containing constructs. However, as we also demonstrate, appropriate folding of kringle structures is essential for angiostatin to maintain its full anti-endothelial activity.

There is now considerable direct evidence that tumor growth is angiogenesis-dependent(1-4). The most compelling evidence is based on the discovery of angiostatin, an angiogenesis inhibitor that selectively instructs endothelium to become refractory to angiogenic stimuli(5). Angiostatin, which specifically inhibits endothelial proliferation, induced dormancy of metastases defined by a balance of apoptosis and proliferation(6). We now show that systemic administration of human angiostatin potently inhibits the growth of three human and three murine primary carcinomas in mice. An almost complete inhibition of tumor growth was observed without detectable toxicity or resistance. The human carcinomas regressed to microscopic dormant foci in which tumor cell proliferation was balanced by apoptosis in the presence of blocked angiogenesis. This regression of primary tumors without toxicity has not been previously described. This is also the first demonstration of dormancy therapy, a novel anticancer strategy in which malignant tumors are regressed by prolonged blockade of angiogenesis.

BACKGROUND:In previous animal studies, interleukin 12 (IL 12) was shown to inhibit the growth of a wide spectrum of tumors in vivo but to have no direct effect on tumor cells in vitro. Also, contrary to the expectation of a T-cell-mediated effect, the antitumor activity of IL 12 was not completely abrogated in tests of T-cell-deficient mice. These observations suggest that IL 12 may possess antiangiogenic properties that account for its tumor-inhibitory effects in vivo.PURPOSE:Our goal was to investigate the hypothesis that IL 12 has antiangiogenic properties.METHODS:A model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL 12 and interferon gamma (IFN gamma) on angiogenesis in vivo. Different strains of male mice, e.g., immunocompetent C57BL/6 mice, severe combined immune-deficient (SCID) mice, natural killer cell-deficient beige mice, and T-cell-deficient nude mice, were treated with IL 12 (1 microgram/day) intraperitoneally for 5 consecutive days. The extent of neovascularization in response to a basic fibroblast growth factor pellet and the inhibition of neovascularization by IL 12 or IFN gamma were assessed by measuring the maximal vessel length and the corneal circumference involved in new blood vessel formation. The antitumor activities of IL 12 and of the angiogenesis inhibitor AGM-1470 were evaluated in Lewis lung carcinoma-bearing mice. In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreatic islet endothelial cells, and mouse hemangioendothelioma cells.RESULTS:IL 12 treatment almost completely inhibited corneal neovascularization in C57BL/6, SCID, and beige mice. This potent suppression of angiogenesis was prevented by the administration of IFN gamma-neutralizing antibodies, suggesting that the suppression was mediated through IFN gamma. In addition, the administration of IFN gamma reproduced the antiangiogenic effects observed during treatment with IL 12. Treatment with IL 12 and AGM-1470 combined did not increase toxicity and showed a trend toward enhanced antitumor efficacy in Lewis lung carcinoma-bearing mice.CONCLUSIONS:IL 12 strongly inhibits neovascularization. This effect is not mediated by a specific cell type of the immune system. Instead, IL 12 has been shown to induce IFN gamma, which, in turn, appears to play a critical role as a mediator of the antiangiogenic effects of IL 12.IMPLICATIONS:Recognition of the mechanisms of the antiangiogenic properties of IL 12 may be crucial in planning its clinical applications, including a possibility of coadministration with other inhibitors of neovascularization.

Hemangioma and other angiomatous diseases of childhood are common. Although most lesions are harmless and self-limiting, some are associated with significant morbidity and may be life-threatening. Interferon-alpha, a weak angiogenesis inhibitor, recently has been found to significantly reduce the mortality rate associated with life-threatening hemangiomas. The effectiveness of AGM-1470, a potent inhibitor of angiogenesis derived from the fungal product fumagillin, was tested in a mouse model of hemangioendothelioma. Thirty syngeneic mice were implanted with cells derived from a spontaneous mouse hemangioendothelioma. Tumors formed within 2 to 3 days, and the animals were then treated systemically with AGM-1470 or with saline and vehicle alone. After 22 days, the tumor volume in the saline-treated mice was 7368 +/- 2723 mm3, versus 709 +/- 73 mm3 in the mice that received AGM-1470 (P < .001). Survival was prolonged for the AGM-1470-treated mice, and there was no evidence of drug-related toxicity. All experiments were repeated. In this study, AGM-1470 was safe and highly effective in the treatment of hemangioendothelioma. AGM-1470, and other antiangiogenic agents, may provide safe and effective treatment for hemangioma and other angiomatous diseases.

In cancer patients, dormant micrometastases are often asymptomatic and clinically undetectable, for months or years, until relapse. We have studied dormant lung metastases under angiogenesis suppression in mice. The metastases exhibited rapid growth when the inhibition of angiogenesis was removed. Tumour cell proliferation, as measured by bromodeoxyuridine incorporation and immunohistochemical staining proliferating cell nuclear antigen, was not significantly different in dormant and growing metastases. However, tumour cells of dormant metastases exhibited a more than threefold higher incidence of apoptosis. These data show that metastases remain dormant when tumour cell proliferation is balanced by an equivalent rate of cell death and suggest that angiogenesis inhibitors control metastatic growth by indirectly increasing apoptosis in tumour cells.

The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a primary tumor inhibits its remote metastases. After tumor removal, metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kDa plasminogen fragment that we have sequenced and named angiostatin. A corresponding fragment of human plasminogen has similar activity. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin.

Ischemia often precedes neovascularization. In ocular neovascularization, such as occurs in diabetic retinopathy, a diffusible angiogenic factor has been postulated to be produced by ischemic retina and to lead to neovascularization of the retina, optic nerve, or iris. However, no angiogenic factor has been conclusively identified that satisfies this hypothesis. Vascular endothelial growth factor/vascular permeability factor, hereafter referred to as VEGF, is a likely candidate for an ocular angiogenic factor because it is a secreted mitogen, specific for endothelial cells, and is upregulated by hypoxia. We investigated the association of VEGF with the development of experimental iris neovascularization in the cynomolgus monkey. Following the production of retinal ischemia by laser occlusion of all branch retinal veins, VEGF was increased in the aqueous fluid, and the aqueous VEGF levels changed synchronously and proportionally with the severity of iris neovascularization. Northern analysis and in situ hybridization revealed that VEGF messenger RNA is upregulated in the ischemic retina. These observations support the hypothesis that ocular neovascularization is regulated by a diffusible factor and identify VEGF as a likely candidate for a retina-derived vascular permeability and angiogenesis factor in vivo.

A central problem in tumor growth is how tumor cells switch to the angiogenic phenotype. Virtually all solid tumors are angiogenic and neovascularized by the time they are detectable in animals and humans. However, at the time they originate, spontaneously arising tumor cells are not usually angiogenic (Folkman et al. 1989). Tumors formed by these cells are of small volumes limited to a few cubic millimeters and restricted to an existence in this in situ stage for months or years. Further expansion of such a tumor depends on its induction of new capillary blood vessels that converge toward the tumor. This switch to angiogenesis is usually accomplished by a subset of tumor cells within the in situ lesion. The new microvessels that are recruited provide a neovascular meshwork which supports the growth, and facilitates invasion and metastasis of the rapidly expanding tumor...