Inhibition of angiogenesis by thrombospondin-1 (A2)

描述(申请人提供)：血栓反应蛋白-1(TSP-1)在与血管生成、伤口愈合和肿瘤形成相关的组织重塑过程中调节内皮细胞表型。观察到TSP-1基因缺失的小鼠肿瘤生长增加，基于TSP-1的治疗方法目前正在进行抑制血管生成的临床试验，这突显了TSP-1抑制血管生成的重要性。拟议的研究集中在以下几个方面。具体目的1.在临床前期小鼠模型中，确定3TSR和TRAIL受体激动剂抗体靶向肿瘤内皮细胞和肿瘤细胞的有效性。TSP-1(3TSR)和肿瘤坏死因子相关的凋亡诱导配体(TRAIL)的三个1型重复序列分别通过诱导内皮细胞和肿瘤细胞凋亡而诱导包括结肠癌在内的多种肿瘤细胞的凋亡。然而，TRAIL和3TSR均不能诱导肿瘤完全消退。我们的研究表明，3TSR可以上调原代培养的人真皮微血管内皮细胞(HDMEC)上的死亡受体(DR4和DR5)，从而使这些正常的TRAIL抵抗细胞对TRAIL诱导的细胞凋亡敏感。此外，3TSR和TRAIL受体激动剂抗体的结合导致在结肠癌小鼠模型中显著抑制肿瘤生长。本研究的目的是(1)建立结肠癌动物模型中CD36、caspase-8和caspase-9的激活以及小鼠DR5(MDR5)的上调来诱导内皮细胞的凋亡，以及(2)在使用结肠癌皮下和原位模型进行的预防和回归/干预治疗试验中，测试使用3TSR使肿瘤内皮细胞对TRAIL诱导的细胞凋亡敏感的组合方法的治疗效果。具体目的2.鉴定介导TSP-1和3TSR抗血管生成活性的受体和信号分子。虽然Fyn、JNK和p38磷酸化已被证明参与TSP-1诱导内皮细胞的凋亡，但该信号转导通路的其余成分尚不清楚。我们发现CD36与血管内皮细胞生长因子受体2(VEGFR2)、整合素、Tetraspanins及其相关的信号转导分子有关。我们推测，内皮细胞膜上的CD36/VEGFR2/整合素复合体介导了3TSR诱导的细胞凋亡，并促进了促血管生成和抗血管生成的信号转导通路之间的串扰。这项研究的目的是确定(1)CD36/VEGFR2/整合素复合体的哪些成分在TSP-1或3TSR诱导的内皮细胞凋亡中是必不可少的；(2)这些复合体是否能够使内皮细胞整合肿瘤微环境中的促血管生成和抗血管生成信号；以及(3)确定TSP-1或3TSR的存在如何影响这些复合体的结构和功能。这些研究将为3TSR和TRAIL联合治疗的潜力提供关键的见解，并将阐明TSP-1抑制血管生成的分子基础。与公共健康相关：这项拟议的研究旨在开发新的策略，通过破坏为肿瘤提供氧气和营养的血管来抑制肿瘤生长。这项拟议研究的目标是确定人体通常用来限制血管生长的蛋白质是如何发挥作用的。这些知识将被用来开发治疗癌症的方法。

DESCRIPTION (provided by applicant): Thrombospondin-1 (TSP-1) regulates endothelial cell phenotype during the tissue remodeling that is associated with angiogenesis, wound healing and neoplasia. The importance of suppression of angiogenesis by TSP-1 is underscored by the observation that TSP-1-null mice exhibit increased tumor growth and that TSP-1-based therapeutics are currently in clinical trials for the inhibition of angiogenesis. The proposed studies focus on the following areas. Specific Aim 1. To determine the effectiveness of targeting tumor endothelial cells and tumor cells with 3TSR and TRAIL receptor agonist antibodies in preclinical mouse models. Others and we have found that the three type 1 repeats of TSP-1 (3TSR) and TNF-Related Apoptosis Inducing Ligand (TRAIL) can induce apoptosis of various tumor cells, including colon carcinomas, through the induction of endothelial cell apoptosis and tumor cell apoptosis, respectively. However, neither TRAIL nor 3TSR can induce complete tumor regression. Our studies have demonstrated that 3TSR can up- regulate death receptor (DR4 and DR5) in primary human dermal microvascular endothelial cells (HDMEC) and this sensitizes these normally TRAIL resistant cells to TRAIL-induced apoptosis. Furthermore, a combination of 3TSR and a TRAIL receptor agonist antibody results in dramatic inhibition of tumor growth in a murine model of colon cancer. The goals of this aim are to (1) establish that induction of endothelial cell apoptosis in colon cancer animal models involves CD36, activation of caspase-8 and -9, and up-regulation of murine DR5 (mDR5) and (2) test the therapeutic efficacy of combinatorial approaches using 3TSR to sensitize tumor endothelial cells to TRAIL-induced apoptosis in prevention and regression/intervention therapeutic trials using subcutaneous and orthotopic models of colon cancer. Specific Aim 2. To identify the receptors and signaling molecules that mediate the anti-angiogenic activity of TSP-1 and 3TSR. Whereas Fyn, JNK and p38 phosphorylation have been shown to be involved in TSP-1-induced endothelial cell apoptosis, the remaining components of this signal transduction pathway are unknown. We have found that CD36 associates with vascular endothelial cell growth factor receptor 2 (VEGFR2), integrins, tetraspanins and their associated signal transduction molecules. We hypothesize that the CD36/VEGFR2/integrin complexes in the endothelial cell membrane mediate 3TSR-induced apoptosis and facilitates cross-talk between pro- and anti-angiogenic signal transduction pathways. The goals of the proposed study are to determine (1) which components of the CD36/VEGFR2/integrin complexes are essential for TSP-1- or 3TSR-induced endothelial cell apoptosis, (2) whether these complexes enable endothelial cells to integrate pro- and anti-angiogenic signals in the tumor microenvironment and (3) determine how the presence of TSP-1 or 3TSR affects the structure and function of these complexes. These studies will provide key insights into the therapeutic potential of 3TSR and TRAIL combined therapy and will elucidate the molecular basis for the inhibition of angiogenesis by TSP-1. PUBLIC HEALTH RELEVANCE: The proposed research is designed to develop new strategies for inhibiting tumor growth by destroying the blood vessels that supply oxygen and nutrients to them. The goal of the proposed study is to determine how proteins that the body normally uses to limit blood vessel growth work. This knowledge will be used to develop therapeutic approaches for the treatment of cancer.