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Contents
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Controlling Angiogenesis Through Thrombospondin-1 Regulation of Nitric Oxide Signaling
One of these is the potent endogenous angiogenesis inhibitor thrombospondin-1, a drug mimetic of which (ABT-510) is currently in phase II clinical trials for cancer treatment. Expression of thrombospondin-1 is commonly diminished or absent in pathology specimens from several major cancers, and studies in mice showed that approximately 0.1 nM levels of circulating thrombospondin-1 can limit tumor growth and angiogenesis. Yet, previous studies using cultured vascular endothelial cells required 1 to 10 nM concentrations of thrombospondin-1 to inhibit their growth or movement. Our collaborative studies have identified crosstalk between NO and thrombospondin-1 in endothelial cells that can explain this discrepancy. In the above, and in an accompanying paper (Ridnour LA et al. Proc Natl Acad Sci U S A 102: 13147–52, 2005), we describe novel mechanisms by which thrombospondin-1 inhibits angiogenesis stimulated by NO. In Isenberg et al., we show that low-dose NO increases the efficacy of thrombospondin-1 to inhibit endothelial cell growth, movement, and adhesion by a factor of 100 to 1000. We show that this activity is shared by antibodies that recognize the thrombospondin-1 receptor CD36 and by recombinant parts of the thrombospondin-1 molecule known to interact with this receptor on endothelial cells. This inhibition is mediated by way of thrombospondin-1 blocking the NO-mediated activation of soluble guanylyl cyclase (sGC). This enzyme mediates the synthesis of cyclic-GMP (cGMP) in cells, an important molecule in signaling pathways leading to tumor angiogenesis (Figure 1, part A). By blocking the NO-mediated activation of sGC, thrombospondin-1 also blocks the ability of an angiogenic molecule produced by many tumors, vascular endothelial growth factor, to stimulate cGMP production in endothelial cells. Finally, using transgenic mice, we show that levels of cGMP in vascular endothelial cells are elevated in the absence of endogenous thrombospondin-1 and are more sensitive to further elevation in response to NO donors. Therefore, endogenous levels of thrombospondin-1 clearly limit NO signaling through this pathway in vascular cells. In Ridnour et al., we show that NO and thrombospondin-1 form a feedback loop, whereby NO downregulates thrombospondin-1 and thrombospondin-1 inhibits NO-stimulated pathways that induce angiogenesis (Figure 1, part B). At low levels of NO (1 nM), thrombospondin-1 expression is blocked at the mRNA and protein levels, facilitating the pro-angiogenic activity of NO. This inhibition is reversed at higher levels of NO via induction of the phosphatase MKP-1, engaging inhibitory feedback to limit the angiogenic response to NO. Finally, at high NO levels such as would be produced by activated macrophages (1 μM), angiogenesis is directly inhibited by NO via phosphorylation of p53. This finely tuned feedback mechanism appears to be critical to control both wound healing and tumor angiogenesis. Figure 1. Cross talk between thrombospondin-1 and nitric oxide (NO) controls angiogenesis. A) Angiogenic signaling induced by vascular endothelial growth factor (VEGF) through its receptor activates Akt, which in turn phosphorylates and activates endothelial nitric oxide synthase (eNOS). The resulting NO binds to and activates soluble guanylyl cyclase (sGC), leading to accumulation of intracellular cyclic-GMP (cGMP). cGMP binds to and activates kinases (cGKs) and cGMP-gated channels (cNG) to stimulate endothelial cell responses required for angiogenesis. Thrombospondin-1 inhibits sGC activation and thereby prevents angiogenic signaling. B) Complementing the blocking of NO signaling by thrombospondin-1, low pro-angiogenic doses of NO suppress thrombospondin-1 expression to remove this inhibitor and facilitate angiogenesis. At higher levels of NO, this feedback is reversed by induction of additional signals that restore expression of inhibitory thrombospondin-1 as well as direct inhibition of angiogenesis by NO-derived reactive nitrogen species. Our ongoing studies suggest that both tumor growth and wound healing processes, such as those secondary to surgical treatment of solid tumors, can be controlled by peptides derived from thrombospondin-1 that target NO signaling mechanisms. Our data and those from other recent publications show that nitric oxide synthase (NOS) inhibitors can increase the efficacy of radiation and chemotherapy. Similarly, ABT-510, the drug mimetic of thrombospondin-1 mentioned earlier, binds to CD36 and enhances tumor responses to radiation and chemotherapy. The identification of this novel relationship between thrombospondin-1 and NO and the molecular mechanisms involved reveals new molecular targets for controlling angiogenic responses and could lead to novel treatment strategies combining these agents to increase cancer survival.
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reventing angiogenesis—the recruitment of new blood vessels—has become a major focus for cancer treatment and prevention. Angiogenesis is tightly regulated by a balance between pro- and antiangiogenic factors. The gaseous redox molecule nitric oxide (NO) is known to play a crucial role in blood pressure control, but it was also recently found to promote angiogenesis at physiological levels. Although the latter activity of NO is beneficial in wound-healing responses, it may also promote angiogenesis in tumors. Several pro- and two antiangiogenic factors have been shown to modulate the endothelial form of an enzyme that generates NO. Now, ongoing studies by our group and that of David Wink, PhD (Radiation Biology Branch), reveal that additional molecular targets involved in redox signaling are convergent nodes for signaling by a variety of antiangiogenic agents. 