? Endothelial cell (EC) adaptation to the complex local hemodynamic environment plays a critical role in both the physiological and pathological regulation of vessel wall biology, but mechanisms by which ECs transduce fluid mechanical forces into biochemical signals remains poorly understood. This proposal will address two key questions related to the initiation of mechanotransduction: is extracellular applied fluid force transmitted to the interior of the cell where biochemical signaling molecules are located, and is flow-induced intracellular deformation concentrated at discrete locations in the cell at a magnitude that mediates structural protein interactions? High-resolution 4-D microscopy imaging of green fluorescent protein fused to vimentin, actin, and paxillin will enable measurements to test the hypothesis that changes in extracellular applied fluid shear stress induce spatially focused mechanical responses in the cytoskeleton near intracellular locations where structural proteins are involved in rapid mechanochemical signal transduction. This hypothesis suggests that strain focusing by local cytoskeletal deformation provides the spatial organization of structural proteins necessary to trigger specific biochemical signaling networks in response to changes in the hemodynamic environment.
The specific aims are (1) to determine the spatiotemporal distribution of strain focusing in the actin microfilament network in living ECs during a change in shear stress, (2) to determine the relative contributions of microfilament and intermediate filament networks in focusing cytoskeletal strain during onset of shear stress, and (3) to determine whether focusing of cytoskeletal strain in response to shear stress occurs near sites of focal adhesion to the extracellular matrix and initiates spatial redistribution of focal adhesion proteins. Since focal adhesion proteins are rapidly phosphorylated by onset of shear stress, signaling at these locations may be initiated by mechanical interactions with the cytoskeleton. A novel measurement of interaction strain will be defined to indicate the degree of structural rigidity of mechanical connections between the cytoskeleton and focal adhesion sites. This proposal will measure for the first time spatial and temporal relationships between mechanical interactions in the cytoskeleton and locations involved in initiation of mechanotransduction. The long-term goal of this research program is to define biomechanical mechanisms contributing to cell and tissue function in order to develop innovative approaches for treating endothelial dysfunction in vascular pathology and artificial graft design. ? ?
