Regulation of angiogenic balance by thrombospondin-1

血小板反应蛋白-1 调节血管生成平衡

• 批准号: 6779791
• 负责人: OLGA Valery VOLPERT
• 金额: $ 29.19万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779791
• 关键词: JUN kinase; angiogenesis; angiogenesis inhibitors; apoptosis; cysteine endopeptidases; enzyme activity; enzyme induction /repression; flow cytometry; fluorescence microscopy; genetic transcription; green fluorescent proteins; human tissue; laboratory mouse; microinjections; neutralizing antibody; polymerase chain reaction; protein structure function; thrombospondins; tissue /cell culture; vascular endothelial growth factors; vascular endothelium

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.

描述（由申请人提供）：血管生成（毛细血管发芽）是 通过诱导因子和抑制因子之间的平衡来调节 内皮细胞环境。在培养物中，血管生成刺激促进内皮细胞 细胞存活，而抑制剂则诱导细胞凋亡。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 在多种人类肿瘤中下调，并阻止或减缓肿瘤生长 实验动物。我们证明 TSP-i 通过诱导细胞凋亡在体内发挥作用 在活化的内皮细胞中。这种细胞凋亡是由一个信号介导的 通过与细胞表面的 CD36 结合而启动（参见初步数据）。 TSP-i 和其他抑制剂仅针对重塑血管。我们的结果表明 TSP-i 诱导细胞凋亡需要 Fas/Fas 配体 (FasL) 二级级联 其中 TSP-i 导致内皮细胞 FasL 增加，而 Fas 则增加 VEGF 激活后上调。下面的实验旨在 进一步阐明负责内皮细胞的分子事件 TSP-i 引起的细胞凋亡和活化内皮细胞在重塑中的敏感性 TSP-i 可以促进血管凋亡和抗血管生成。我建议： 1.研究JNK-1和JNK-2与TSP-i诱导细胞凋亡的相关性。 表达 JNK-1 及其显性干扰突变体的构建体 激活激酶SEK-1将被引入内皮细胞和TSP-i 测量诱导信号传导和细胞凋亡。 JNK-1 和 JNK-2 无效的小鼠将 测试支持 TSP-i 的抗血管生成活性的能力。 2.定义 TSP-i 诱导细胞凋亡所必需的半胱天冬酶并按顺序排列。 抗体、荧光底物和特异性抑制剂将定时使用 检测 TSP-i 处理的内皮细胞中激活的半胱天冬酶的研究， 识别重要的信号，并将它们按顺序排列在 TSP-i 诱导的信号传导中 级联。 3.定义内皮细胞上调FasL的介质。这 p38 JNK-1 和 caspase 对转录水平和表面的影响 FasL 的呈现将使用特定的抑制剂和显性的抑制剂来确定 阴性突变体。 Fas对转录水平和激活状态的影响 激酶和半胱天冬酶将使用中和抗 fas 来测定 抗体。 4.寻找利用Fas-FasL二级级联的抗血管生成分子 阻断血管生成和使内皮细胞敏感的血管生成刺激 抑制剂通过 Fas 上调。血管抑制素、内皮抑素、 2-甲氧基酯二醇和 PEDF 将用于诱导 EC 细胞凋亡 体外抗 Fas 中和抗体并阻断小鼠血管生成 Fas 或 FasL 缺陷。将测试 VEGF、bFGF、IL-8 的能力 通过培养物中的内皮细胞和体内的新毛细血管增加表面 Fas 小鼠角膜和/或基质胶塞。 我希望了解靶内皮细胞如何变得敏感 TSP-i 和其他天然存在的血管生成抑制剂导致细胞凋亡 将有助于设计化合物来提高那些有前途的药物的功效或 以保护新形成的脉管系统。这种方法有可能引导 临床试验旨在单独使用其最有效的用途，并结合 常规化疗，并用于预防，延缓肿瘤的进行性生长 休眠的肿瘤。