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.
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