Integrin-Laminin Interactions in Endothelial Cells

描述（由申请人提供）：新血管的建立，一个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进展。我们最近的工作提供了支持的概念，α 4层粘连蛋白亚基（α 4 LN），由内皮细胞分泌的基底膜的组成部分，是至关重要的血管发育，通过一种新的相互作用与α v β 3整联蛋白。该提案有三个目标。在目的1中，将通过使用重组蛋白和经典的生物化学分析或通过相位展示技术来表征α 4 LN亚基的G结构域内的整联蛋白识别基序。在第二个目标中，将评估调节α v β 3整联蛋白对α 4 LN亚基的亲和力的分子机制。这一目的是基于一系列初步数据，表明1）在内皮细胞中含有整合素和α v β 3整合素异源二聚体之间存在串扰，2）这种串扰调节α v β 3整合素对α 4 LN亚基的亲和力，3）PKA抑制剂消除串扰。基于这些数据，我们将确定α v β 3整联蛋白或可能的α v β 3整联蛋白相关蛋白的胞质尾部上的特定丝氨酸/苏氨酸磷酸化事件是否负调节α v β 3整联蛋白对α 4LN的亲和力。目的3是基于表明细胞凋亡在Matrigel上体外内皮细胞的管状网络组装中起作用的研究，并且这种调节的细胞凋亡事件可以被过量的α 4LN配体抑制。为了进一步分析，将在体外评价内皮细胞在管状网络组装过程中调节细胞凋亡的作用。为此，将在内皮细胞的体外形态发生期间用细胞凋亡标记物对内皮细胞进行染色，以评估在管组装期间细胞凋亡在空间上发生的位置。此外，还将评估α v β 3-整联蛋白相互作用在使用阻断或活化配体和整联蛋白抗体、肽激动剂和拮抗剂以及通过分子遗传学方法形成管期间调节内皮细胞存活途径的能力。总之，这些结果将为调节血管生成的分子机制提供新的见解，并且从长远来看，可能会发现有用的试剂，这些试剂可以通过抑制其发展血管系统的能力来阻断肿瘤的发展。

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.