EXTRACELLULAR MATRIX AS A SOLID STATE REGULATOR IN ANGIOGENESIS

这项续订提案的总体目标仍然是理解 细胞外基质(ECM)调节血管发育的生物力学机制。 在上一次资助期间，我们使用了微米尺度上包含定义大小和形状的ECM涂层粘附岛的微型制造基板，以证明ECM基于其抵抗细胞牵引力、改变细胞骨架(CSK)中的张力，从而调节细胞形状的能力，调控毛细血管内皮细胞(CE)是否会生长、分化、经历凋亡或迁移，以及对血管生成因子的反应。研究发现，CSK张力依赖于收缩微丝、微管和ECM粘连之间的机械相互作用，并需要整合素受体与CSK之间通过结合局部黏附蛋白(如纽蛋白)进行有效的机械偶联。这种物理连接的维持也被发现是小G蛋白RAC刺激的片脂形成所必需的，其中，由于药物抑制CSK张力的产生，片脂运动迅速被抑制。因此，本提案的主要目的是扩展我们正在进行的研究，并将重点放在ECM的生物力学机制上：通过ECM调节血管生成刺激的方向迁移和血管构型。研究的具体目的包括：1)探讨细胞外基质结构和力学参数的变化如何影响CSK的张力和局灶性黏附的形成；2)确定CE细胞和ECM之间的机械相互作用如何控制膜脂的伸展和定向迁移；3)探讨在毛细血管形态发生过程中，CSK张力的局部变化是否有助于图案的形成。

The general goal of this renewal proposal continues to be to understand the biomechanical mechanism by which extracellular matrix (ECM) regulates capillary development. During the last grant period,we used microfabricated substrates containing ECM-coated adhesive islands of defined size and shape on the micronscale to demonstrate that ECM governs whether capillary endothelial(CE) cells will grow, differentiate, undergo apoptosisor migratein response to angiogenic factors based on its ability to resist cell tractional forces, alter tension in the cytoskeleton (CSK), and thereby modulate cell shape. CSK tensionwas found to be dependent on mechanical interactions between contractile microfilaments, microtubules, and ECM adhesions and to require efficient mechanical coupling between integrin receptors and the CSK through binding of focal adhesion proteins,such as vinculin. Maintenance of this physical linkage also was found to be required for formation of lamellipodia stimulated by the small G protein, rac, where as pharmacological inhibition of CSK tension generation rapidly suppressed lamellipodial movement. Thus, the main objective of the present proposal is to extend our ongoing studies and to focus on the biomechanical mechanism bywhich ECM: regulates direction almigration and vascular patterning in response to angiogenic stimuli. The specificaims include: 1) to explore how changes in ECM structure and mechanicsalter CSK tensionand focal; adhesion formation,2) to determine how mechanical interactions between CE cellsand ECM control amellipodia extension and directional migration, and 3) to explore whether local changes in CSK tension contribute to pattern formation during capillary morphogenesis.