Regulation of angiogenic balance by thrombospondin-1

描述(申请人提供)：血管生成(毛细血管发芽)是 通过诱导和抑制因子之间的平衡来调节 内皮细胞环境。在培养中血管生成刺激促进内皮细胞生长 当抑制剂诱导细胞凋亡时，细胞存活。凝血酶敏感蛋白-1(TSP-I)，a 有效的血管生成抑制因子，是一种分泌的大分子 在几种人类肿瘤中下调，并阻断或减缓肿瘤的生长 实验动物。我们发现TSP-I在体内通过诱导细胞凋亡发挥作用。 在激活的内皮细胞中。这种细胞凋亡是由一种信号介导的， 是由其与细胞表面的CD36结合启动的(见初步数据)。 TSP-I和其他抑制剂只针对血管重塑。我们的结果表明 TSP-I诱导的细胞凋亡需要Fas/Fas配体(FasL)的第二级联反应 其中TSP-I引起内皮细胞FasL增加，而Fas 经血管内皮生长因子激活后上调。下面的实验旨在 进一步阐明与内皮细胞有关的分子事件 TSP-I诱导的细胞凋亡与活化内皮细胞在重塑中的敏感性 TSP-I对血管细胞凋亡和抗血管生成的影响。我建议： 1.探讨JNK-1和JNK-2在TSP-I诱导细胞凋亡中的作用。 表达JNK-1及其显性干扰突变体的构建 激活激酶SEK-1将被引入内皮细胞和TSP-I 检测诱导信号和细胞凋亡。JNK-1和JNK-2基因缺失的小鼠将 检测支持TSP-I抗血管生成活性的能力。 2.对TSP-I诱导细胞凋亡所必需的caspase进行定义和排序。 抗体、荧光底物和特定的抑制剂将用于Timed 检测经TSP-I处理的血管内皮细胞中激活的caspase的研究， 确定必要的基因，并将它们按顺序排列在TSP-I诱导的信号中 卡斯卡德。 3.明确内皮细胞上调FasL的介质。这个 P38JNK-1和半胱氨酸天冬氨酸蛋白酶对转录水平和表面的影响 FasL的呈递将使用特定的抑制物和优势 阴性变种人。Fas对转录水平和激活状态的影响 将使用中和抗FAS来确定激酶和半胱氨酸天冬氨酸酶的 抗体。 4.寻找利用Fas-FasL次级级联作用的抗血管生成分子 阻断血管生成和血管生成刺激，使内皮细胞对 抑制物通过Fas上调。血管抑素，内皮抑素， 2-甲氧基雌二醇和PEDF将用于诱导EC在有 抗Fas中和抗体的体外实验及阻断小鼠血管生成的实验研究 Fas或FasL缺乏。将检测血管内皮生长因子、碱性成纤维细胞生长因子、白介素8的能力 培养的血管内皮细胞和新生毛细血管增加表面Fas 小鼠角膜和/或Matrigel塞子。 我希望了解靶血管内皮细胞是如何致敏的 TSP-I和其他天然血管生成抑制剂对细胞凋亡的影响 将有助于设计化合物来提高那些有希望的试剂或 以保护新形成的血管系统。这种方法有可能引导 单独进行临床试验，以期达到最有效的使用，与 常规化疗，并用于预防，以延缓渐进性生长 潜伏的肿瘤。

DESCRIPTION (provided by applicant): 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.