EXTRACELLULAR MATRIX AS A SOLID STATE REGULATOR IN ANGIOGENESIS

这项延期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 和发展。在上一批资助期内完成的工作 证实了细胞骨架(CSK)的张力依赖性变化 组织结构和细胞形态在细胞周期进程中起着关键作用 毛细血管内皮细胞。这些研究还显示， 形成分子桥梁的焦点黏附复合体(FAC) 整合素和CSK是信号整合的主要部位 来自生长因子和ECM，以及一条首选的途径 机械应力在细胞表面的传递。因此，主要的 这项建议的目的是分析生物力学机制。 通过ECM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。至 要做到这一点，我们将使用上一篇中开发的微图案化技术 允许我们诱导形成FACS的授权期 大小、形状和位置。微图案化表面将用于 结合细胞外基质的免疫荧光控制。 微操作技术将与显微荧光技术结合使用，以 探讨机械应力是否应用于整合素和 通过FAC传播可以改变S所需的核功能 阶段进入，如核运输。微图案化的表面和 将使用分离FAC的生化方法来确定如何 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。 (例如，TNP-470、TIMPs、Angiostatin、Endostatin)对Fac结构和 信令功能。这种方法应该会加深我们对 血管生成调控的分子基础。它还可以促进 可应用于临床的新型血管生成抑制剂的设计 未来的审判。

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.