Interaction of thrombospondin-1 with alpha9beta1 integrin in glioma angiogenesis

描述（由申请人提供）：先前的研究表明，在内皮细胞上表达的某些整合素在血管生成的进展中起重要作用，并且是开发可用于治疗各种肿瘤的血管抑制药物的有吸引力的靶点。 使用免疫组织化学，我们检测了α 9 <$1整合素在身体所有器官中形成毛细血管的内皮细胞上的表达，以及其在某些肿瘤（包括胶质瘤）中的水平上调。 然而，在分离后，培养物中的大多数原代内皮细胞停止表达α 9 β 1整联蛋白。 A9的灵敏度1整合素在体外内皮细胞上的表达是研究人员从未认为这种整合素是调节新血管形成过程的重要受体的原因，特别是在肿瘤发生期间。 在拟议的研究计划中，我们将探讨在脑肿瘤血管形成的背景下，这种整合素与血小板反应蛋白-1（TSP-1）相互作用后在血管生成中的作用。 最近，我们评估了TSP-1是α 9 <$1整合素的配体，α 9 <$1整合素在这种细胞外基质蛋白的N-末端（NoC 1）结构域上具有结合位点。 基于先前发表的工作，TSP-1在脑肿瘤进展过程中上调，我们提出了一个普遍的假设，即α 9 <$1与TSP-1的相互作用是扩散性胶质瘤发展过程中发生的病理性血管生成的重要因素。 为了验证这一假设，我们提出了一系列的体外和体内实验，可能会导致解释α 9 β 1整合素和TSP-1的作用，促进病理血管生成诱导脑肿瘤。 我们将分离出A9 1整联蛋白阳性的原发性神经胶质瘤人微血管内皮细胞（gHMVEC），其来自手术后获得的癌组织，通过在表达该整联蛋白和典型的内皮细胞标志物如CD 31的第一代细胞中进行免疫分选。 我们将研究TSP-1及其重组NoC 1结构域诱导的这些细胞的促血管生成活性，如增殖和迁移，并评估在这些过程中细胞内激活的信号通路。 我们将使用从正常脑和不同级别星形胶质细胞衍生肿瘤获得的石蜡切片的双荧光颜色免疫组织化学证实我们的预期，即在胶质瘤进展期间，α 9 <$1与TSP-1的相互作用广泛地发生在内皮细胞上。 在动物实验中，我们打算证明通过特异性单克隆抗体或MLD-去整合素VLO 5阻断α 9 <$1整合素将通过阻断血管化过程来抑制实验性胶质瘤的发展。 此外，我们将用NoC 1结构域转染神经胶质瘤细胞，我们期望在将这些转染子颅内植入大鼠后观察到更高的肿瘤生长率。 在研究的另一部分，我们将进行NoC 1结构域的结构/功能分析，以定位TSP-1上的α 9 <$1整合素结合位点。 这项工作将通过化学合成跨越N-末端模块的NoC 1结构域的肽，并通过定点诱变的重组片段的TSP-1的这一部分。 我们将使用特异性单克隆抗体2D 11在Western blot中识别NoC 1，研究临床和实验胶质瘤组织中NoC 1样结构域与正常脑组织相比的上调。 此外，肿瘤NoC 1样结构域的结构表征将使用蛋白质组学方法进行。 公共卫生相关性：胶质瘤是最常见和最难治疗的脑肿瘤之一。 该肿瘤属于血管化最多的癌症，血管抑制治疗将阻断病理组织中的血管生长，似乎在其治疗中是有效的。 在这种情况下，我们提出了一个调查的受体，α 9 <$1整合素，是目前的内皮细胞，这是在血管壁的主要结构细胞。 这种受体的研究可能对癌症以及患有心血管疾病的患者有益，因为血管形成过程的调节在这些病理学中是重要的。

DESCRIPTION (provided by applicant): Previous studies revealed that certain integrins expressed on endothelial cells play a significant role in the progression of angiogenesis and are an attractive target for the development of angiostatic drugs that may have an application in the therapy of various tumors. Using immunohistochemistry we detected the expression of a9¿1 integrin on endothelial cells forming blood capillaries in all organs of the body, and an up-regulation of its level in certain tumors including gliomas. However, following isolation the majority of primary endothelial cells in the culture stop expressing a9¿1 integrin. This sensitivity of a9¿1 integrin on endothelial cells in vitro was the reason that researchers never considered this integrin as an important receptor for modulation of the neovascularization process, especially during oncogenesis. In the proposed research plan we will investigate a role for this integrin in angiogenesis following its interaction with thrombospondin-1 (TSP-1), in the context of brain tumor vascularization. Recently, we evaluated that TSP-1 is a ligand for a9¿1 integrin, which has a binding site on the N-terminal (NoC1) domain of this extracellular matrix protein. Based on previously published work that TSP-1 is up-regulated during brain tumor progression, we proposed a general hypothesis that the interaction of a9¿1 with TSP-1 is an important element of pathological angiogenesis occurring during diffusive glioma development. To verify this hypothesis we propose a series of experiments in vitro and in vivo that may lead to an explanation of a9¿1 integrin's and TSP-1's role in the promotion of pathological angiogenesis induced in brain tumors. We will isolate a9¿1 integrin-positive primary glioma human microvascular endothelial cells (gHMVEC) from cancer tissue obtained following surgery, by immuno-sorting in first passage cells expressing this integrin and typical endothelial cell markers such as CD31. We will investigate pro-angiogenic activities of these cells such as proliferation and migration, induced by TSP-1 and its recombinant NoC1 domain, as well as evaluate the signaling pathway that is activated inside the cell in these processes. We will confirm our expectation that the interaction of a9¿1 with TSP-1 extensively occurs on endothelial cells during glioma progression using double fluorescent color immunohistochemistry of paraffin sections obtained from normal brain and different grades of astrocyte-derived tumors. In animal experiments, we intend to prove that the blocking of a9¿1 integrin by a specific monoclonal antibody or by a MLD-disintegrin, VLO5 will suppress the development of experimental glioma by blocking the vascularization process. Also, we will transfect glioma cells with the NoC1 domain and we expect to observe a higher ratio of tumor growth following intracranial implantation of these transfectants into rats. In another part of the study, we will perform a structure/function analysis of the NoC1 domain to localize the a9¿1 integrin binding site on TSP-1. This work will be performed by chemical synthesis of peptides spanning the N-terminal module of the NoC1 domain and by site-directed mutagenesis of recombinant fragments of this part of TSP-1. We will investigate the up-regulation of NoC1-like domain in clinical and experimental glioma tissues in comparison with normal brain using a specific monoclonal antibody, 2D11 that recognizes NoC1 in Western blot. Further, the structural characterization of tumoral NoC1-like domain will be performed using proteomic approaches. PUBLIC HEALTH RELEVANCE: Glioma is one of the most frequently occurring and difficult to treat brain tumors. This tumor belongs to the most vascularized cancers and angiostatic treatment, which will block vessel growth in pathological tissue, appears to be effective in its therapy. In this context, we propose an investigation a receptor, a9¿1 integrin that is present on the endothelial cells, which are the major structural cells in vessel wall. Investigation of this receptor may be beneficial for cancer as well as for cardiovascular disease having patients, because regulation of vascularization process is important in these pathologies.