This revised competing renewal application for BRP EB 02185 (formerly HL 64381) responds to PAR 04- 023. An integrative systems approach is used to translate concepts from basic science rooted in the biomechanics of leukocyte adhesion to applications of molecular imaging using targeted ultrasound contrast agents. As proposed in the NIH roadmap document, in this application """"""""scientists move beyond the confines of their own discipline and explore new organizational models for team science"""""""". This collaborative, interdisciplinary application has one basic science aim and three design-directed, applied aims. In the previous funding period, we measured the force per single bond of P- and L-selectin over a wide range of loading rates using a laser trap. We studied the cellular biomechanics of leukocytes as they become adherent in flow chamber systems and in postcapillary venules in vivo. Rolling leukocytes exhibit specialized surface structures (tethers) and whole-cell deformations that form the basis for modifying our molecularly targeted ultrasound contrast agents to improve their attachment under flow. Antibodies to P-selectin, alphaVbetaS integrin, and targeting peptides were conjugated to lipid-shelled microbubbles and successfully applied to image tumor angiogenesis and inflammation of the kidneys, heart, and other tissues in mice. Now, we propose to expand the basic science studies, optimize the contrast agents, and take the molecular ultrasound contrast technology into the apoE-/- mouse model of atherosclerosis to image vulnerable atherosclerotic plaque. This is highly relevant to human disease, because up to 40% of all myocardial infarctions are caused by the rupture of vulnerable plaques that are not visible by angiographic methods. Preliminary data show that our improved contrast agents can be used to image atherosclerotic plaque. To develop the best possible ligands, we propose a ligand core facility at the University of Virginia that will produce, purify and couple various suitable targeting molecules to contrast agents.
The specific aims are to (1) further define the cellular and molecular biomechanics of leukocyte adhesion under flow conditions with specific emphasis on measuring 2D molecular on-rates and the role of deformation in stabilizing adhesion; (2) determine the biomechanical properties of microbubble-based, targeted ultrasound contrast agents and to modify these properties for optimal adhesion under flow in vitro and in vivo, using the principles gleaned from leukocyte adhesion studies; (3) test how multiple ligands with different molecular properties attached to ultrasound contrast agents can provide improved targeting specificity and/or efficiency in vitro and in vivo; (4) apply the design developed in aims 2 and 3 to molecular ultrasound imaging of atherosclerotic plaque. Since ultrasound is the most economical of the modern imaging modalities, cost savings in health care can also be expected from the proposed research. ? ? ?
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