This application proposes interdisciplinary bioengineering research in the area of molecular biomechanics. Leukocyte and endothelial adhesion molecules govern the trafficking of cells in inflammation, immunity, cancer mestastasis and other processes. Some adhesion molecules, among them the selectins, are specialized to mediate adhesion in the presence of blood flow. Pressure-driven blood flow is associated with a shear stress exerted on the vessel wall, which results in a force on leukocytes and other cells trying to adhere to the endothelium. It is believed that adhesion under shear stress requires adhesion molecules with rapid association rates (on-rates), resulting in rapid formation of bonds. In vitro experiments and modeling studies indicate that the selectins also have high rates of bond dissociation (off-rates). Preliminary data suggest that the off-rates of selectins vary systematically with the shearing force exerted on the cell bound by the selectin (reactive compliance or tensile strength). In addition, the release of at least one of the selectins is accelerated by proteolytic cleavage by a surface-bound or membrane integral metalloproteinase. The current proposal has four specific aims. (1) To measure the bond lifetimes and apparent off-rates of L-, P- and E-selectin bound to their natural ligands. (2) To determine the role of L-selectin shedding in regulating leukocyte adhesion and selectin kinetics. (3) To determine the impact of the selectin length and their cytoplasmic tail for the biomechanics of adhesion under shear flow. (4) To design and build beads, liposomes and gas-filled bubbles (ultrasound contrast agents) that use leukocyte adhesion molecules to bind to vessel walls under shear stress. Each of these aims is approached in a three-pronged fashion. We propose to use laser trapping technology to directly measure biomechanical and kinetic parameters of selectin bonds, use single cells on sparse substrates to understand the biomechanics of selectins in an in vitro flow chamber, and use intravital microscopy to study selectin biomechanics in the context of the living organism. We propose to use molecular biology techniques to manipulate cDNA, cells, and mice to isolate each molecular mechanism. We will use the insights gained to design liposome-based targeted drug delivery systems and ultrasound contrast microbubbles for delivery in the vascular system under shear flow. At the end of the first year, we plan to have measurements of selectin off-rates, taking into account selectin shedding, and have tested selectin-containing liposomes for their ability to adhere under shear. Milestones for the following years are listed in the timeline.
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