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理解将有助于组织工程和治疗心血管疾病。生物材料科学的一个特别令人兴奋的领域是设计可以诱导细胞粘附，扩散和细胞间通讯的材料。在这里，我们开发了一种新的方式来切换“开”和“关1的底物的构象，通过组装肽配体在水凝胶表面上使用卷曲螺旋肽组件诱导的小分子活化剂，如阳离子。粘附肽和生长因子肽将使用激活剂浓度的变化连接到水凝胶表面。将使用牵引力显微镜（TFM）评估细胞响应表面活化的行为，其中对细胞施加在表面上的力进行成像。我们将联合收割机和TFM结合起来，测量内皮细胞的粘附、扩散和细胞间相互作用，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，我们将开发使用卷曲螺旋肽结构域在表面组装肽的方法。我们将组装的肽是RGD，在纤连蛋白的细胞结合域中的肽; PHSRN，纤连蛋白协同位点;表皮生长因子（EGF）;和血管内皮生长因子（VEGF）。VEGF和VEGF是明显的选择，分别涉及细胞粘附增强和血管生成。在目标2中，我们将研究粘附肽和生长因子肽的协同递送如何诱导单个内皮细胞的伸展和力产生。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作为肽类型，肽浓度，时间和底物顺应性的函数的细胞扩展和力的产生。我们将专注于配体对，使用RGD作为共同的肽，并将其与协同位点或生长因子肽相结合。在目标3中，我们将通过测量作为两种内皮细胞类型的配体类型、配体密度、时间和底物顺应性的函数的细胞粘附概率或分散度，来测量粘附和生长因子配体的组合如何诱导内皮细胞之间的细胞-细胞通信。最后，我们将使用我们的肽表面来测试“差异粘附假说”，其中细胞-细胞通信可以通过改变细胞基质粘附来工程化。

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.