Integrin-Laminin Interactions in Endothelial Cells

描述（由申请人提供）：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进展。我们最近的工作支持了a4层粘连蛋白亚基（alpha4LN）的概念，即内皮细胞分泌的基底膜的一个组成部分，通过与α β 3整合素的新相互作用对血管发育至关重要。这项提议有三个目的。在目标1中，整合素识别基序(s)在alpha4LN亚基的G结构域将使用重组蛋白和经典的生化分析或相显示技术进行表征。在第二个目标中，将评估调节alphabeta3整合素对alpha4LN亚基亲和力的分子机制。这一目的是基于一系列的初步数据，这些数据表明：1)在内皮细胞中含有整合素的(1)与α β 3整合素异源二聚体之间存在串扰；2)这种串扰调节α β 3整合素对（4LN亚基的亲和力；3）PKA抑制剂消除了这种串扰。基于这些数据，我们将确定在alphavbeta3整合素的细胞质尾部的特定丝氨酸/苏氨酸磷酸化事件，或者可能是alphavbeta3整合素相关蛋白，是否负向调节alphavbeta3整合素对alpha4LN的亲和力。Aim 3的基础研究表明，细胞凋亡在体外基质上内皮细胞的管状网络组装中起作用，并且这种受调节的凋亡事件可以通过过量的α 4ln配体来抑制。为了进一步分析，我们将在体外评估内皮细胞在管状网络组装过程中调控凋亡的作用。为此，内皮细胞将在体外形态形成过程中被细胞凋亡标志物染色，以评估细胞凋亡在试管组装过程中的空间位置。此外，将通过阻断或激活配体和整合素抗体、肽激动剂和拮抗剂以及分子遗传学方法，评估α - β -整合素相互作用在管形成过程中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，从长远来看，可能会发现有用的试剂，可以通过抑制肿瘤形成血管系统的能力来阻止肿瘤的发展。

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.