Molecular Mechanism of Endothelial Migration Under Flow

DESCRIPTION (provided by the applicant): The vascular endothelial cells (ECs) play important roles in the regulation of vascular functions, including cell adhesion, inflammatory responses, vasoactivity, and macromolecular permeability. The loss of endothelial integrity can lead to thrombosis, atherosclerosis, and stenosis, and EC migration in wound healing is required for the restoration of its integrity and functions. Although EC migration has been studied extensively, there is relatively little information on the effects of flow on EC migration and the underlying molecular mechanisms. EC migration involves dynamic and coordinated changes in cytoskeletal organization, signal transduction, and cell adhesions to neighboring cells and extracellular matrices (ECM). In this proposal, we will determine the effects of shear stress, the tangential component of hemodynamic forces, on the dynamics and interactions of cytoskeleton, EC-ECM adhesion, and cell-cell adhesion. Our hypothesis is as follows: EC migration is the net result of the following factors: (1) Intracellular factors (e.g., cytoskeleton and signaling events), (2) EC-ECM adhesion, (3) ECEC adhesion, and (4) Externally applied forces such as shear stress. Shear stress can induce intracellular mechanochemical transduction to modify the dynamics and force balance in EC-ECM and EC-EC adhesions, thus modulating EC migration and wound healing. In order to test our hypothesis, three Specific Aims are proposed. (1) To determine the roles of EC-ECM adhesion and shear stress in focal adhesion dynamics, cytoskeletal remodeling, and force generation on ECM during the migration of subconfluent ECs. (2) To elucidate the roles of Rho family GTPases in focal adhesion dynamics, cytoskeletal remodeling, and force generation on ECM during the migration of subconfluent ECs. (3) To determine the roles of cell-cell coupling in modulating EC migration in the healing of confluent monolayer after wounding. The experiments will be conducted with an interdisciplinary approach, including the tracking of the dynamics of cytoskeletal and adhesion proteins with green fluorescence protein, the monitoring of cell migration under well-defined shear stress in a flow chamber, the manipulation of signaling molecules with negative and active mutants, and the computation of traction force exerted by migrating cells on ECM with the use of the newly developed beads-in-membrane method. Since the vascular tree is constantly exposed to flow forces, the proposed research on the effects of flow on EC migration will generate important new information relevant to many physiological processes and pathophysiological conditions.