Regulation of angiogenic balance by thrombospondin-1

描述（由申请人提供）：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调节， 内皮细胞环境。在培养物中，血管生成刺激物促进内皮细胞 细胞存活，而抑制剂诱导凋亡。血小板反应蛋白-1（TSP-i），a 血管生成的有效抑制剂，是一种大的分泌分子， 在几种人类肿瘤中下调，并阻断或减缓肿瘤生长， 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。这种凋亡是由一种信号介导的， 通过与细胞表面上的CD36结合而启动（见初步数据）。 TSP-i和其他抑制剂仅靶向重塑血管。我们的研究结果表明 TSP-i诱导的细胞凋亡需要Fas/Fas配体（FasL）二级级联反应 其中TSP-i引起内皮细胞的FasL增加，而Fas是 在其被VEGF激活后上调。以下实验旨在 进一步阐明了负责内膜细胞的分子事件 TSP-i诱导的细胞凋亡与活化内皮细胞对重塑的敏感性 TSP-i对血管凋亡和抗血管生成的影响。我提议： 1.探讨JNK-1和JNK-2与TSP-1诱导的细胞凋亡的相关性。 表达JNK-1及其受体的显性干扰突变体的构建体 激活激酶SEK-1将被导入内皮细胞和TSP-1中， 诱导的信号传导和测量的凋亡。JNK-1和JNK-2缺失的小鼠将 测试支持TSP-1的抗血管生成活性的能力。 2.定义并排列TSP-1诱导的细胞凋亡所必需的半胱天冬酶。 抗体，荧光底物和特异性抑制剂将被定时使用。 检测TSP-1处理的内皮细胞中激活的半胱天冬酶的研究， 确定必需的，并将它们按顺序放置在TSP-1诱导的信号传导中 级联。 3.定义内皮细胞上调FasL的介质。的 p38 JNK-1和caspase对转录水平和表面活性的影响 FasL的表达将使用特异性抑制剂和显性抑制剂来确定。 阴性突变体Fas对转录水平和激活状态的影响 激酶和半胱氨酸天冬氨酸蛋白酶的活性将使用中和性抗Fas抗体测定 抗体的 4.寻找利用Fas-FasL二级级联反应的抗血管生成分子， 阻断血管生成和血管生成刺激， 抑制剂通过上调Fas的表达。血管抑素，内皮抑素， 2-将使用间氧雌二醇和PEDF在存在下诱导EC凋亡。 抗Fas中和抗体在体外和阻断小鼠血管生成 Fas或FasL缺乏。将测试VEGF、bFGF、IL-8的能力， 通过培养中内皮细胞和新生毛细血管增加表面Fas 小鼠角膜和/或基质胶塞。 我希望了解靶内皮细胞是如何变得敏感的， TSP-i和其他天然存在的血管生成抑制剂引起的细胞凋亡 将有助于设计化合物，以提高这些有前途的药物的功效， 以保护新生血管这种方法有可能引导 临床试验，以达到最有效的单独使用，结合 常规化疗，并用于预防，以延缓进行性生长， 休眠的肿瘤

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.