Interaction of thrombospondin-1 with alpha9beta1 integrin in glioma angiogenesis

血小板反应蛋白-1 与 α9β1 整合素在胶质瘤血管生成中的相互作用

• 批准号: 8288605
• 负责人: CEZARY MARCINKIEWICZ
• 金额: $ 30.19万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288605
• 关键词: Adhesions; Amino Acid Sequence; Amino Acids; Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Angiogenesis Modulating Agents; Animal Experiments; Animal Model; Antibodies; Autopsy; Baculoviruses; Binding; Binding Sites; Biological Assay; Blood capillaries; Brain; Brain Neoplasms; C-terminal; Cardiovascular Diseases; Cell Proliferation; Cell Survival; Cell membrane; Cells; Clinical; Color; Development; Digestion; Disintegrins; Elements; Embryo; Endothelial Cells; Endothelium; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Extracellular Matrix Proteins; Family; Fluorescein; Gene Silencing; Glioblastoma; Glioma; Goals; Growth; Health; Human; Immunohistochemistry; Immunoprecipitation; Implant; In Vitro; Integrin Binding; Integrins; Investigation; Label; Laboratories; Lead; Ligands; Malignant Neoplasms; Modeling; Monoclonal Antibodies; Mutate; Mutation; N-terminal; Nature; Operative Surgical Procedures; Organ; PECAM1 gene; Paraffin; Pathologic Neovascularization; Pathology; Patients; Peptides; Pharmaceutical Preparations; Physiological; Play; Process; Proteins; Proteomics; Publishing; Quail; Radial; Rattus; Recombinants; Regulation; Reporting; Research; Research Personnel; Role; Series; Signal Pathway; Site; Site-Directed Mutagenesis; Small Interfering RNA; Snake Venoms; Sorting - Cell Movement; Structure; Subfamily lentivirinae; System; Testing; Thrombospondin 1; Tissues; Tumor Angiogenesis; Up-Regulation; Vascularization; Western Blotting; Work; analog; angiogenesis; astrocyte-derived tumor; base; brain tissue; cell motility; expectation; extracellular; glioma cell line; implantation; in vivo; inhibitor/antagonist; integrin alpha9beta1; migration; mutant; neovascularization; peptide chemical synthesis; receptor; research study; synthetic peptide; tumor; tumor growth; tumor progression; tumorigenesis

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.

描述(申请人提供)：先前的研究表明，内皮细胞上表达的某些整合素在血管生成的进程中发挥着重要作用，是开发可能应用于各种肿瘤治疗的血管抑制药物的一个有吸引力的靶点。利用免疫组织化学方法，我们检测到A9？1整合素在体内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，培养中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考虑将其作为调节新生血管过程的重要受体的原因，尤其是在肿瘤形成过程中。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，它在这种细胞外基质蛋白的N端(Noc1)区域有一个结合位点。基于先前发表的TSP-1在脑肿瘤进展过程中上调的研究，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生发展过程中病理性血管生成的重要因素。为了验证这一假说，我们提出了一系列体外和体内实验，可能有助于解释A9？1整合素和TSP-1‘S在促进脑肿瘤病理性血管生成中的作用。我们将从手术后获得的癌组织中分离出A9？1整合素阳性的原发胶质瘤人微血管内皮细胞(GHMVEC)，方法是在表达这种整合素的第一代细胞和典型的内皮细胞标记物(如CD31)中进行免疫分选。我们将研究这些细胞的促血管生成活性，如TSP-1及其重组NoC1结构域诱导的增殖和迁移，以及在这些过程中细胞内激活的信号通路。我们将通过对正常脑组织和不同级别的星形细胞肿瘤的石蜡切片进行双荧光彩色免疫组织化学染色，证实我们的预期，即A9？1与TSP-1在胶质瘤进展过程中广泛存在于内皮细胞上。在动物实验中，我们打算证明，用特定的单抗或MLD去整合素VLO5阻断A9？1整合素，将通过阻断血管形成过程来抑制实验性脑胶质瘤的发展。此外，我们还将把NoC1域的胶质瘤细胞导入大鼠体内，并期望观察到这些转基因细胞在大鼠脑内植入后有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域N-末端模块的多肽和通过对TSP-1这一部分的重组片段进行定点突变来进行。我们将使用一种在Western印迹中识别NoC1的特异性单抗2D11，与正常脑组织相比，研究临床和实验胶质瘤组织中NoC1样结构域的上调。此外，肿瘤NoC1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：胶质瘤是最常见和最难治疗的脑肿瘤之一。该肿瘤属于血管生成最多的肿瘤，血管抑制治疗将阻断病理组织中的血管生长，在其治疗中似乎是有效的。在这种背景下，我们建议研究一种受体，A9？1整合素，它存在于内皮细胞上，内皮细胞是血管壁的主要结构细胞。对这种受体的研究可能对癌症和心血管疾病患者有益，因为血管形成过程的调节在这些病理中很重要。

项目成果

Non-RGD-containing snake venom disintegrins, functional and structural relations.

不含RGD的蛇毒解整合素，功能和结构关系。

• DOI: 10.1016/j.toxicon.2011.07.004
• 发表时间: 2011
• 期刊: Toxicon : official journal of the International Society on Toxinology
• 作者: Walsh,ErinM;Marcinkiewicz,Cezary
• 通讯作者: Marcinkiewicz,Cezary

Vipegitide: a folded peptidomimetic partial antagonist of α2β1 integrin with antiplatelet aggregation activity.

• DOI: 10.2147/dddt.s72844
• 发表时间: 2015
• 期刊: Drug design, development and therapy
• 作者: Momic T;Katzhendler J;Shai E;Noy E;Senderowitz H;Eble JA;Marcinkiewicz C;Varon D;Lazarovici P
• 通讯作者: Lazarovici P

alpha(9)beta(1) integrin engagement inhibits neutrophil spontaneous apoptosis: involvement of Bcl-2 family members.

• DOI: 10.1016/j.bbamcr.2010.03.012
• 发表时间: 2010-07
• 期刊: Biochimica et biophysica acta
• 作者: R. Saldanha-Gama;J. Moraes;Andréa Mariano-Oliveira;A. L. Coelho;E. Walsh;C. Marcinkiewicz;C. Barja-Fidalgo