The general goal of this proposal is to understand the mechanism by which extracellular matrix (ECM) molecules regulate capillary endothelial (CE) cell sensitivity to soluble mitogens, such as FGF. The design of this proposal is based on studies carried out in the past funding period which demonstrate that ECM molecules, such as fibronectin (FN), control CE cell growth based on a """"""""mechanochemical"""""""" mechanism, that is, by resisting cell tension and thereby promoting large-scale changes of cell and nuclear shape. We now propose to directly measure mechanical forces that are transmitted across cell surface ECM receptors (integrins) and throughout the cytoskeletal-nuclear matrix network in order to begin to translate cell shape control of growth into molecular and biophysical terms. We also will determine whether mechanically-induced changes of nuclear structure alter nuclear functions that are required for cell cycle progression, such as nuclear protein transport. CE cell shape and growth will be controlled by varying cell-FN contact formation using a method devised in the past grant period. Cytofluorimetry and 3H-thymidine autoradiography will be carried out to map restriction points during cell cycle progression that are sensitive to cell shape and/or changes in cytoskeletal organization. Cell magnetometry will be used to directly measure cytoskeletal tension and viscoelasticity within living cells and to relate these mechanical properties to effects on cell shape, nuclear size, and growth. Electron microscopy, fluorescence microscopy, and computerized image analysis will then be used to determine whether changes in cytoskeletal tension, produced either by altering cell-FN contact formation or physically deforming cells, can alter nuclear pore size and/or nuclear transport rates. Probes to be tested will include colloidal gold particles and fluorescent proteins that are conjugated to synthetic peptides containing the putative nuclear translocation sequence that has been reported to mediate cell cycle- dependent transport of FGF into the nucleus. Finally, studies will be carried out to analyze the mechanism of action of a novel angiogenesis inhibitor (AGM-1470) which we discovered during the past grant period because it induced cell rounding and which is currently entering Phase I clinical trials for the treatment of cancer. In this manner, we hope to better understand the mechanism by which angiogenesis is controlled in the local tissue microenvironment and thereby facilitate development of new and improved therapeutic modalities for the treatment of cancer.
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