The goal of this proposal is to characterize the molecular mechanism by which extracellular matrix (ECM) regulates capillary endothelial (CE) cell growth during angiogenesis. While the growth control field has focused on early integrin and growth factor signaling events, studies carried out in the past grant period revealed that ECM-dependent changes in cell shape and cytoskeletal structure play an equally critical role in control of cell cycle progression. Specifically, growth factor- stimulated CE cells must spread on ECM and maintain an intact actin cytoskeleton in order to upregulate cyclin D1, down regulate the cdk inhibitor, P27kip1 (p27) and thus, pass through the late G1/S restriction point. These effects on cyclin D1 and p27 appear to be exerted largely at the translational level. In separate studies, we found that mechanical distortion of the cell and cytoskeleton caused by cell-ECM binding alters the organization of the cell's protein translational machinery, as indicated by tension-dependent recruitment of mRNA and ribosomes to the focal adhesion. Thus, the specific aims of this continuation proposal are: 1) to use transfection and biochemical techniques to demonstrate that the effects of cell shape on G1 progression are mediated through changes in cyclin D1 and/or p27 protein levels, 2) to use transfection and microinjection techniques to determine whether the small G protein, Rho, mediates the effects of cell shape on p27 and/or cyclin D1, and 3) to use in situ hybridization and biochemical analysis of UTR reporter constructs to analyze how distortion of the cell and cytoskeleton alters translation of p27 and cyclin D1. Cell shape will be controlled independently of the total cell-ECM binding area using microfabricated adhesive islands that exhibit defined size and geometry on the micron scale. Controlled stresses will be applied to integrins and cytoskeleton using magnetic twisting cytometry. This experimental approach will enable us to begin to translate the long recognized coupling between cell shape and growth into molecular terms. It also may lead to identification of novel molecular targets for angiogenesis inhibition and hence, new approaches to anti-cancer therapy.
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| Mammoto, Tadanori; Parikh, Samir M; Mammoto, Akiko et al. (2007) Angiopoietin-1 requires p190 RhoGAP to protect against vascular leakage in vivo. J Biol Chem 282:23910-8 |
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