Force Microscopy of Endothelial Cells on Novel Peptide Materials

描述(由申请人提供)：对生物细胞如何与材料相互作用的基本了解将有助于组织工程和治疗心血管疾病。生物材料科学中一个特别令人兴奋的领域是可以诱导细胞黏附、扩散和细胞间通讯的材料的设计。在这里，我们开发了一种新的方法来打开和关闭底物的粘附性，通过使用由小分子激活剂(如阳离子)诱导的盘状多肽组件在水凝胶表面组装多肽配体来实现。粘附肽和生长因子多肽将通过改变激活剂浓度连接到球杆上的水凝胶表面。细胞对表面激活的反应将使用牵引力显微镜(TFM)来评估，在TFM中，细胞施加在表面上的力被成像。我们将结合我们的粘合技术和TFM来测量内皮细胞的黏附、扩散和细胞与细胞的相互作用，内皮细胞是心血管系统中参与血管止血和血管生成的关键细胞。在目标1中，我们将开发利用卷曲的多肽结构域在表面组装多肽的方法。我们将组装的多肽是RGD，纤维连接蛋白细胞结合区域的多肽；PHSRN，纤维连接蛋白协同位点；表皮生长因子(EGF)；以及血管内皮生长因子(VEGF)。EOF和VEGF是明显的选择，它们分别与细胞黏附增强和血管生成有关。在目标2中，我们将研究粘附肽和生长因子多肽的协同传递如何诱导单个内皮细胞的扩散和力的产生。我们将测量两种类型的内皮细胞：牛主动脉内皮细胞和微血管内皮细胞的细胞扩散和力的生成，作为多肽类型、多肽浓度、时间和底物顺应性的函数。我们将关注配基对，使用RGD作为共同的多肽，并将其与协同位点或生长因子多肽结合。在目标3中，我们将通过测量两种内皮细胞类型、配体密度、时间和底物顺应性的函数来测量细胞的黏附概率或分散度，以衡量黏附和生长因子配体的组合如何诱导内皮细胞之间的细胞间通讯。最后，我们将使用我们的多肽表面来测试“差异黏附假说”，在该假说中，细胞与细胞之间的通讯可以通过改变细胞底物黏附来实现。

DESCRIPTION (provided by applicant): A fundamental understanding of how biological cells interact with materials would be useful for tissue engineering, and treating cardiovascular disease. A particularly exciting area of biomaterials science is the design of materials that can induce cell adhesion, spreading, and cell-cell communication. Here, we develop a new way of switching "on" and "off1 the adhesiveness of the substrate, by assembling peptide ligands on a hydrogel surface using coiled-coil peptide assemblies induced by small molecule activators, such as cations. Adhesive peptides and growth factors peptides will be linked to a hyrdogel surface on cue, using a change in activator concentration. The behavior of cells in response to surface activation will be assessed using traction force microscopy (TFM), in which the forces exerted by cells on surfaces are imaged. We will combine our adhesive technology and TFM to measure the adhesion, spreading, and cell-cell interactions of endothelial cells, a critical cell in the cardiovascular system involved in blood vessel hemostasis and angiogenesis. In aim 1, we will develop methods to assemble peptides at surfaces using coiled-coil peptide domains. Peptides we will assemble are RGD, the peptide in the cell binding domain of fibronectin; PHSRN, the fibronectin synergy site; epidermal growth factor (EGF); and vascular endothelial growth factor (VEGF). EOF and VEGF are obvious choices which have implicated in cell adhesion strengthening and angiogenesis, respectively. In aim 2, we will study how the coordinated delivery of adhesive and growth factor peptides can induce the spreading and force generation of single endothelial cells. We will measure cell spreading and force generation as a function of peptide type, peptide concentration, time, and substrate compliance, for two types of endothelial cells : bovine aortic endothelial cells and microvascular endothelial cells. We will focus on pairs of ligands, using RGD as the common peptide, and combining it with either the synergy site or a growth factor peptide. In aim 3, we will measure how the combination of adhesive and growth factor ligands can induce cell-cell communication between endothelial cells, by measuring the adhesion probability or the dispersion of cells as a function of ligand type, ligand density, time, and substrate compliance for both endothelial cell types. Finally, we will use our peptide surfaces to test the "differential adhesion hypothesis", in which cell-cell communication can be engineered through alteration in cell substrate adhesion.