Interaction of thrombospondin-1 with alpha9beta1 integrin in glioma angiogenesis

描述（由申请人提供）：以往的研究表明，在内皮细胞上表达的某些整合素在血管生成的进程中起着重要作用，是开发血管抑制药物的一个有吸引力的靶点，可能在各种肿瘤的治疗中有应用。利用免疫组化技术，我们检测了a9¿1整合素在人体所有器官中形成毛细血管的内皮细胞上的表达，并在包括胶质瘤在内的某些肿瘤中表达上调。然而，在分离后，培养的大多数原代内皮细胞停止表达a9¿1整合素。a9¿1整合素在体外对内皮细胞的敏感性是研究人员从未将其视为调节新血管形成过程的重要受体的原因，特别是在肿瘤发生过程中。在拟议的研究计划中，我们将在脑肿瘤血管形成的背景下，研究这种整合素在与血栓反应蛋白-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a9¿1整合素的配体，该整合素在该细胞外基质蛋白的n端（NoC1）结构域上有一个结合位点。基于先前发表的研究成果，TSP-1在脑肿瘤进展过程中被上调，我们提出了一个普遍的假设，即a9¿1与TSP-1的相互作用是弥漫性胶质瘤发展过程中病理性血管生成的重要因素。为了验证这一假设，我们提出了一系列体外和体内实验，这些实验可能会解释a9¿1整合素和TSP-1在促进脑肿瘤诱导的病理性血管生成中的作用。我们将从手术后获得的癌症组织中分离出a9¿1整合素阳性的原发性胶质瘤人微血管内皮细胞（gHMVEC），通过免疫分选表达该整合素和典型内皮细胞标记物（如CD31）的首次传代细胞。我们将研究由TSP-1及其重组NoC1结构域诱导的这些细胞的增殖和迁移等促血管生成活性，并评估在这些过程中细胞内激活的信号通路。我们将证实我们的预期，a9¿1与TSP-1的相互作用在胶质瘤进展过程中广泛发生在内皮细胞上，使用从正常大脑和不同级别的星形胶质细胞来源的肿瘤中获得的石蜡切片的双荧光免疫组织化学。在动物实验中，我们打算证明通过特异性单克隆抗体或mvd -解体素VLO5阻断a9¿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NoC1结构域转染胶质瘤细胞，我们期望在大鼠颅内植入这些转染物后观察到更高的肿瘤生长比率。在研究的另一部分中，我们将对NoC1结构域进行结构/功能分析，以定位TSP-1上的a9¿1整合素结合位点。这项工作将通过化学合成跨越NoC1结构域n端模块的肽，以及对TSP-1这部分的重组片段进行定点诱变来完成。我们将使用特异性单克隆抗体2D11在Western blot中识别NoC1，研究临床和实验脑胶质瘤组织中NoC1样结构域与正常脑相比的上调。此外，肿瘤no1样结构域的结构特征将使用蛋白质组学方法进行。公共卫生相关性：神经胶质瘤是最常见和最难治疗的脑肿瘤之一。这种肿瘤属于血管化程度最高的癌症，血管抑制治疗可以阻断病理组织中的血管生长，似乎对其治疗有效。在这种情况下，我们建议研究一种受体，即存在于内皮细胞上的9¿1整合素，内皮细胞是血管壁的主要结构细胞。这种受体的研究可能对癌症和心血管疾病患者有益，因为血管化过程的调节在这些病理中很重要。

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.