Angiogenesis, (capillary sprouting) is regulated via balance between inducing and inhibitory factors in the endothelial cell environment. In culture angiogenic stimuli promote endothelial cell survival while inhibitors induce apoptosis. Thrombospondin- 1 (TSP-i), a potent inhibitor of angiogenesis, is a large secreted molecule that is down-regulated in several human tumors and blocks or slows tumor growth in experimental animals. We showed that TSP-i acts in vivo by inducing apoptosis in the activated endothelial cells. This apoptosis is mediated by a signal that is initiated by its binding to CD36 on the cell surface (see preliminary data). TSP-i and other inhibitors target only remodeling vessels. Our results indicate that TSP-i induced apoptosis requires Fas/Fas ligand (FasL) secondary cascade where TSP-i causes increase of the FasL by endothelial cells while Fas is up-regulated upon their activation by VEGF. Experiments below are designed to further elucidate molecular events responsible for the endotheial cell apoptosis by TSP-i and the sensitivity of activated endotheium in remodeling vessels to the apoptosis and anti-angiogenesis by TSP-i. I propose to: 1.Investigate the relevance of JNK- 1 and JNK-2 to TSP-i induced apoptosis. Constructs expressing dominant interfering mutants of JNK- 1 and of its activating kinase SEK- 1 will be introdiced into endothelial cells and TSP-i induced signaling and apoptosis measured. Mice null for JNK- 1 and JNK-2 will be tested for the ability to support anti-angiogenic activity of TSP-i. 2.Define and place in order caspases essential to TSP-i induced apoptosis. Antibodies, fluorogemc substrates and specific inhibitors will be used in timed studies to detect caspases activated in TSP-i treated endotheial cells, identify essential ones, and place them in order in the TSP-i induced signaling cascade. 3.Define mediators of up-regulation of the FasL by endothelial cells. The effect of p38 JNK- 1 and caspases on transcription levels and surface presentation of FasL will be determined using specific inhibitors and dominant negative mutants. The effect of Fas on transcription level and activation state of kinases and caspases will be determined using neutralizing anti-fas antibodies. 4.Seek anti-angiogenic molecules that utilize Fas - FasL secondary cascade to block angiogenesis and angiogenic stimuli that sensitize endothelial cells to the inhibitors via Fas up regulation. Angiostatin, endostatin, 2-metoxyestardiol and PEDF will be used to induce EC apoptosis in the presence of anti-Fas neutralizing antibodies in vitro and to block angiogenesis in mice deficient for Fas or FasL. VEGF, bFGF, IL-8 will be tested for the ability to increase surface Fas by endothelial cells in culture and by new capillaries in mouse cornea and/or Matrigel plugs. It is my hope that understanding how target endothelial cells become sensitized to apoptosis by TSP-i and other naturally occurring inhibitors of angiogenesis will help to devise compounds to improve efficacy of those promising agents or to protect newly forming vasculature. This approach has the potential to steer clinical trials towards their most effective use alone, combined with conventional chemotherapy, and for prevention, to delay progressive growth of the dormant tumors.
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