Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium-derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium-derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-␣-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies. © 2002 Wiley-Liss, Inc. Key words: tumor angiogenesis; thrombospondin-1; peptide mimetics; bladder cancer; melanomaSolid tumors and their metastases are critically dependent on neovascularization for progressive growth. If vessels cannot form, tumors remain dormant at a threshold size of several millimeters in diameter. 1,2 Whether or not vessels develop is determined by the balance between inhibitors of angiogenesis and angiogenic stimuli present within a given tissue microenvironment. In most healthy adult tissues, inhibitors predominate, favoring vascular quiescence, while in many disease states the situation is reversed, with a prevalence of inducers triggering capillary remodeling and angiogenesis. 3,4 During tumorigenesis, downregulation of secreted antiangiogenic mediators is often a key step in the development of an angiogenic phenotype (reviewed in refs. 3-5).Blocking tumor progression by restoring the physiologic antiangiogenic environment was first proposed as an anticancer therapy by Folkman 6 and has been extensively validated in animal models. [7][8][9][10][11][12][13] One potential adva...

During disease progression in the prostate, production of TSP-1, the major inhibitor, is down-regulated while that of stimulatory FGF-2 and/or VEGF rise, leading to the induction of the new vessels necessary to support tumor growth.

Thrombospondin 1 (TSP1) is a multifunctional protein able to activate TGF␤ and to inhibit angiogenesis in vivo. Although usually thought of as an inhibitor of tumor growth, TSP1 may sometimes be present at high levels during tumor progression, suggesting that tumors can eventually overcome their anti-tumor effects. Using a tet-repressible expression system, we demonstrate that murine TSP1 delayed the onset of tumor growth when produced in the tumor bed by rat fibrosarcoma tumor cells or by stromal fibroblasts coinjected with unmodified C6 glioma tumor cells. Yet upon prolonged exposure to TSP1, tumors came to grow at the same rate in the presence as in the absence of TSP1 and transplantation experiments showed that they had become insensitive to inhibition by TSP1 in both syngeneic and immune compromised hosts. Tumor resistance to TSP1 developed as a result of the in vivo outgrowth of pre-existing tumor cell variants that (1) secreted increased amounts of angiogenic factors that counterbalanced the inhibitory effect of TSP1 on neovascularization and (2) grew more efficiently in the presence of TSP1-activated TGF␤. These results indicate that prolonged and continuous local delivery of a single multifunctional angiogenesis inhibitor like TSP1 to fast-growing tumors can lead to tumor resistance in vivo by fostering the outgrowth of subpopulations that are a by-product of the genetic instability of the tumor cells themselves.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.