EXTRACELLULAR MATRIX AS A SOLID STATE REGULATOR IN ANGIOGENESIS

这一更新提案的总体目标仍然是 了解细胞外基质（ECM）如何调节毛细血管生长 发展先行者的要求 在上一个资助期内完成的工作 证明了细胞骨架（CSK）的张力依赖性变化 组织和细胞形状在细胞周期进程中起关键作用， 毛细血管内皮细胞。 这些研究还表明， 粘着斑复合物（FAC），在 整合素和CSK代表整合信号的主要位点 从生长因子和ECM以及一个优选的途径， 机械应力在细胞表面的传递。 因此， 本研究的目的是分析其生物力学机制 ECM通过其促进细胞、CSK和核结构的变化， 特别强调对FAC结构和功能的控制。 到 要做到这一点，我们将使用微图案技术开发的最后一个 允许我们诱导形成具有定义的FACs的授予期 大小形状和位置 微图案表面将用于 结合ECM免疫荧光控制。 显微操作技术将与显微荧光测定法一起使用， 探索是否机械应力施加到整联蛋白和 通过FAC传输可以改变S 阶段进入，如核运输。 微图案化表面和 分离FACs的生化方法将用于确定 来自ECM的信号调节CE细胞对这些有丝分裂原的敏感性。 最后，我们将分析已知的血管抑制化合物的作用 (e.g., TNP-470、TIMP、血管抑素、内皮抑素）对FAC结构的影响， 信号功能。 这一做法应有助于我们了解 血管生成调控的分子基础。 它也可以促进 设计新的血管生成抑制剂， 审判在未来。

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.