Integrin-Laminin Interactions in Endothelial Cells

内皮细胞中整合素-层粘连蛋白的相互作用

• 批准号: 7221242
• 负责人: ANNETTE M GONZALEZ
• 金额: $ 11.89万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7221242
• 关键词: Adhesions; Affinity; Agonist; Amino Acid Sequence; Apoptosis; Apoptotic; Basement membrane; Behavior; Biochemical; Blood Vessels; CD47 Antigen; Cell Adhesion; Cell Surface Receptors; Cell Survival; Cell physiology; Cells; Complex; Condition; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Tail; Data; Development; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Event; Extracellular Matrix Proteins; Family; Goals; Immunoblotting; In Vitro; Integrin Binding; Integrin alphaVbeta3; Integrins; Laminin; Ligands; Ligation; Measures; Mediating; Molecular; Molecular Genetics; Morphogenesis; Numbers; Nutrient; Oligopeptides; PKA inhibitor; Pathway interactions; Peptide Mapping; Peptide antibodies; Peptides; Phage Display; Phase; Phospho-Specific Antibodies; Phosphorylation; Play; Principal Investigator; Process; Proteins; Proteomics; Reagent; Recombinant Proteins; Research; Role; Screening procedure; Series; Serine/Threonine Phosphorylation; Signal Transduction; Staging; Staining method; Stains; Techniques; Technology; Testing; Time; Tube; Work; angiogenesis; base; in vitro Assay; insight; interest; matrigel; migration; mutant; neoplastic cell; novel; programs; tumor; tumor progression

DESCRIPTION (provided by applicant): The establishment of new blood vessels, a process known as angiogenesis, promotes tumor progression by providing tumor cells the necessary nutrients and signals for survival. Our recent work has provided support for the notion that the a4 laminin subunit (alpha4LN), a component of the basement membrane secreted by endothelial cells, is crucial for blood vessel development through a novel interaction with alphavbeta3 integrin. There are three aims in the proposal. In aim 1, integrin recognition motif(s) within the G domain of the alpha4LN subunit will be characterized by using recombinant proteins and classical biochemical analyses or by phase display technique. In the second aim, the molecular mechanisms regulating the affinity of alphavbeta3 integrin for the alpha4LN subunit will be evaluated. This aim is based on a series of preliminary data indicating 1) that there is crosstalk between (1containing integrins and the alphavbeta3 integrin heterodimer in endothelial cells 2) that such crosstalk regulates the affinity of alphavbeta3 integrin for the (4LN subunit and 3) a PKA inhibitor abrogates the crosstalk. Based on these data, we will determine whether specific serine/threonine phosphorylation events on the cytoplasmic tails of alphavbeta3 integrin or, possibly an alphavbeta3 integrin associated protein, negatively regulate the affinity of alphavbeta3 integrin for alpha4LN. Aim 3 is based on studies which indicate that apoptosis plays a role in tube-like network assembly of endothelial cells in vitro on Matrigel and that such regulated apoptotic events can be inhibited by an excess of alpha4LN ligand. To further this analysis, the role of regulated apoptosis during tube-like network assembly by endothelial cells will be evaluated in vitro. To do so, endothelial cells will be stained with markers for apoptosis during their morphogenesis in vitro to assess where apoptosis occurs spatially during tube assembly. In addition, the ability of alphavbeta3-integrin interactions to regulate endothelial cell survival pathways during tube formation using blocking or activating ligand and integrin antibodies, peptide agonists and antagonists, and by molecular genetic approaches will be assessed. Together these results will provide new insight into the molecular mechanisms that regulate angiogenesis and, long term, may uncover useful reagents that can block tumor development by inhibiting their ability to develop a vasculature.

描述(申请人提供)：新血管的建立，一个被称为血管生成的过程，通过为肿瘤细胞提供生存所需的营养和信号来促进肿瘤的进展。我们最近的工作支持了A4层粘连蛋白亚单位(Alpha4LN)是内皮细胞分泌的基底膜的组成部分，通过与Alphavbeta3整合素的新的相互作用对血管发育至关重要的观点。该提案有三个目标。在目标1中，将使用重组蛋白和经典生化分析或相显示技术来表征α4LN亚基G结构域中的整合素识别基序(S)。在第二个目标中，将评估调节αvbeta3整合素与α4LN亚基亲和力的分子机制。这一目的是基于一系列初步数据表明：1)在(1)含有整合素的内皮细胞中存在串扰；2)这种串扰调节αvbeta3整合素与(4LN亚基)的亲和力；3)PKA抑制剂消除了串扰。基于这些数据，我们将确定αvbeta3整合素细胞质尾部的特定丝氨酸/苏氨酸磷酸化事件是否负面调节alphavbeta3整合素与α4LN的亲和力。目的3是基于一些研究表明，细胞凋亡在体外Matrigel上的内皮细胞管状网络组装中发挥作用，并且这种调节的凋亡事件可以被过量的α4LN配体抑制。为了进一步进行这一分析，我们将在体外评估内皮细胞在管状网络组装过程中受调控的细胞凋亡的作用。为了做到这一点，在体外的形态发生过程中，将用内皮细胞的凋亡标志物进行染色，以评估在管子组装过程中，细胞凋亡发生在空间上的哪里。此外，还将评估通过阻断或激活配体和整合素抗体、多肽激动剂和拮抗剂，以及通过分子遗传学方法，α-β3-整合素相互作用在管子形成过程中调节内皮细胞生存途径的能力。总而言之，这些结果将为调节血管生成的分子机制提供新的见解，并从长远来看，可能会发现有用的试剂，可以通过抑制肿瘤血管形成的能力来阻止肿瘤的发展。