FRACTALKINE: ROLES IN CELL ADHESION AND ATHEROSCLEROSIS

本提案的总体目标是确定fractalkine在血管疾病中的作用。Fractalkine是一种在活化内皮细胞上表达的新型趋化因子，最近的数据表明，它可以高亲和力地结合表达Fractalkine受体CX3CR1的细胞。因此，与其他趋化因子不同，fractalkine似乎不仅作为趋化剂起作用，而且作为粘附分子。fractalkine的细胞粘附性能可能是由于其独特的结构。几乎所有其他已知的趋化因子都是分泌的蛋白质。相反，fractalkine是一种跨膜结构域蛋白，其趋化因子样结构域位于粘蛋白柄的顶部。我们最近发现fractalkine捕获在生理相关剪切应力下流动的细胞非常迅速和高效。在Specific Aim 1中，我们将使用一种新的细胞粘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粘附。在Specific Aim 2中，我们将确定fractalkine中的结构域，这些结构域对于介导对表达cx3cr1的细胞的高亲和力粘附至关重要。我们将创造新的嵌合体，其中其他趋化因子取代fractalkine的趋化因子样结构域。使用在Specific Aim 1下开发的分析，我们将确定fractalkine独特的细胞结合特性是由于在刚性茎的顶部呈现趋化因子样结构域还是趋化因子本身的独特特性。在Specific Aim 3中，我们将创建CX3CR1敲除小鼠来直接评估fractalkine在血管疾病中的作用。我们将把这些小鼠培育成适当的遗传背景，以验证fractalkine在两种需要从快速流动的血液中捕获白细胞的人类疾病（动脉粥样硬化和肾小球肾炎）中起重要作用的假设。这项拨款中提出的实验将使用新颖的体外定量分析方法，并创建fractalkine受体敲除小鼠，以提供fractalkine在血管疾病中作用的重要新信息。

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.