FRACTALKINE: ROLES IN CELL ADHESION AND ATHEROSCLEROSIS

这项提案的总体目标是确定Fractalkine在血管疾病中的作用。Fractalkine是一种表达于激活的内皮细胞上的新型趋化因子，最近的研究表明，它与表达Fractalkine受体CX3CR1的细胞具有高亲和力。因此，与其他趋化因子不同，Fractalkine似乎不仅是一种趋化因子，而且还是一种黏附分子。Fractalkine的细胞黏附特性可能与其独特的结构有关。事实上，所有其他已知的趋化因子都是分泌型蛋白质。相比之下，Fractalkine是一种跨膜区蛋白，其趋化素样结构域位于粘蛋白茎的顶部。我们最近发现，Fractalkine捕捉在生理相关剪应力下流动的细胞非常迅速和高效。在具体目标1中，我们将使用一种新的细胞黏附实验来定量比较表达CX3CR1的细胞与Fractalkine的结合与整合素介导的细胞黏附。在具体目标2中，我们将确定Fractalkine中对介导与CX3CR1表达细胞的高亲和力黏附至关重要的结构域。我们将创造新的嵌合体，其中其他趋化因子取代了Fractalkine的趋化素样结构域。使用在特定目标1下开发的分析方法，我们将确定Fractalkine独特的细胞结合特性是由于刚性茎顶部出现趋化素样结构域，还是由于趋化因子本身的独特性质。在具体目标3中，我们将创建CX3CR1基因敲除小鼠，以直接评估Fractalkine在血管疾病中的作用。我们将把这些小鼠培育成合适的遗传背景，以检验这一假设，即Fractalkine在两种人类疾病中发挥重要作用，这两种疾病需要从快速流动的血液中捕获白细胞：动脉粥样硬化和肾小球肾炎。这项资助中提议的实验将使用新颖的、定量的体外分析和建立Fractalkine受体基因敲除小鼠，以提供有关Fractalkine在血管疾病中的作用的重要新信息。

The overall goal of this proposal is to determine the role of fractalkine in vascular disease. Fractalkine is a novel chemokine expressed on activated endothelial cells and recent data indicates that it binds cells expressing the fractalkine receptor, CX3CR1, with high affinity. Thus, unlike other chemokines, fractalkine appears to function not only as a chemoattractant, but also as an adhesion molecule. The cell adhesion properties of fractalkine may be due to its unique architecture. Virtually all other known chemokines are secreted proteins. In contrast, fractalkine is a transmembrane domain protein with a chemokine-like domain located at the top of a mucin stalk. We have recently found that fractalkine captures cells flowing under physiologically relevant shear stress extremely rapidly and with high efficiency. In Specific Aim 1, we will use a novel cell adhesion assay to quantitatively compare the binding of CX3CR1-expressing cells to fractalkine with integrin-mediated cell adhesion. In Specific Aim 2, we will identify domains within fractalkine that are critical for mediating high-affinity adhesion to CX3CR1-expressing cells. We will create novel chimeras in which other chemokines are substituted for the chemokine-like domain of fractalkine. Using the assays developed under Specific Aim 1, we will determine whether the unique cell-binding properties of fractalkine are due to the presentation of the chemokine-like domain at the top of a rigid stalk or to unique properties of the chemokine itself. In Specific Aim 3, we will create CX3CR1 knockout mice to directly assess the role of fractalkine in vascular disease. We will breed these mice into appropriate genetic backgrounds to test the hypothesis that fractalkine plays an important role in two human diseases that require the capture of leukocytes from rapidly flowing blood: atherosclerosis and glomerulonephritis. The experiments proposed in this grant will use novel, quantitative in vitro assays and the creation of a fractalkine receptor knockout mouse to provide significant new information on the role of fractalkine in vascular disease.