The general goal of this RENEWAL proposal continues to be to understand how extracellular matrix (ECM) regulates capillary growth and development. Work complete in the last grant period demonstrated the tension-dependent changes in cytoskeletal (CSK) organization and cell shape play a key role in cell cycle progression in capillary endothelial (CE) cells. These studies also revealed that the focal adhesion complex (FAC) that forms a molecular bridge between integrins and the CSK represents a major site for integration of signals from growth factors and ECM as well as a preferred pathway for transfer of mechanical stresses across the cell surface. Thus, the main objective of this proposal is to analyze the biomechanical mechanism by which ECM promotes changes in cell, CSK, and nuclear structure, with special emphasis on control of FAC structure and function. To do this, we will use a micropatterning technique developed in the last grant period that permits us to induce formation of FACs with defined size, shape, and position. Micropatterned surfaces will be used in conjunction with immunofluorescence control by ECM. Micromanipulation techniques will be used with microfluorimetry to explore whether mechanical stresses applied to integrins and transmitted across the FAC can alter nuclear functions required for S phase entry, such as nuclear transport. Micropatterned surfaces and a biochemical method for isolating FACs will be used to determine how signals from ECM modulates CE cell sensitivity to these mitogens. Finally, we will analyze the effects of known angiostatic compounds (e.g., TNP-470, TIMPs, angiostatin, endostatin) on FAC structure and signaling functions. This approach should further our understanding of the molecular basis of angiogenic regulation. It also may facilitate design of new angiogenesis inhibitors that could be moved into clinical trials in the future.
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