Micromechanical Characterization of Endothelial Cortex

本研究的长期目标是了解细胞力学与血管内皮屏障功能之间的关系。内皮通透性的改变在血管生成、关节硬化和炎症等过程中很重要。血管内皮的细胞骨架是高度动态的，被认为是细胞力学的主要决定因素。此外，细胞骨架的变化已被证明直接影响内皮屏障。这里提出的假设是，这种细胞骨架重组导致局部机械特性的重塑，这反过来又会对屏障功能产生重要影响。因此，我们建议努力表征模型内皮细胞（牛肺动脉细胞）皮层的局部机械结构，并研究这些细胞的机械重塑。为了实现这一目标，我们在此提出了一个试点项目，以开发必要的成像方法来可视化皮质力学，并在细胞骨架和皮质力学结构之间建立明确的联系。这个试点项目有两个具体目标。首先，我们将使用原子力显微镜生成活内皮细胞皮层局部机械结构的高分辨率图像。这些图像将揭示这些细胞的皮层是如何组织的，以及它如何随着时间的推移而重塑。我们还将通过压痕测量来表征局部力学，并将这些测量与皮层形态联系起来。其次，我们将通过使用生化标记和药理学方法确定决定血管内皮细胞机械结构的分子成分。用肌动蛋白、微管蛋白和其他细胞骨架成分特异性抗体标记的细胞共聚焦显微镜之间的相关成像将用于确定在AFM图像中看到的机械特征是由什么细胞结构引起的。此外，药理学药物将用于破坏特定结构，特别是细胞骨架结构，并将其与机械特征的丧失联系起来。本项目的顺利完成，为完整研究血管内皮的皮质力学以及正常和病变细胞的力学重构提供了必要的技术和科学基础。

The long-term goal of the proposed research is to understand the relationship between cell mechanics and barrier functions of the vascular endothelium. Changes in the permeability of endothelia are important in processes such as angiogenesis, arthrosclerosis and inflammation. The cytoskeleton of vascular endothelium is known to be highly dynamic and is thought to be the primary determinant of cellular mechanics. Further, changes in the cytoskeleton have been shown to directly affect the endothelial barrier. The hypothesis put forth here is that such cytoskeletal reorganization results in a remodeling of the local mechanical properties, which in turn will have important consequences for barrier function. Thus, we propose an effort to characterize the local mechanical architecture of the cortex of a model endothelial cell (Bovine Pulmonary Artery Cells), and study the mechanical remodeling of these cells. To achieve this goal we here propose a pilot project to develop the imaging methods necessary to visualize cortical mechanics and to establish a clear connection between the cytoskeleton and cortical mechano-architecture. This pilot project has two specific aims. First we will use atomic force microscopy to produce high-resolution images of the local mechanical architecture of the cortex in living endothelial cells. These images will reveal how the cortex of these cells is organized, and how it remodels with time. We will also characterize the local mechanics through indentation measurements, and relate these measurements to the cortical morphology. Second, we will determine the molecular components that determine the mechanical architecture of vascular endothelial cells by using biochemical labeling and pharmacological approaches. Correlated imaging between confocal microscopy of cells labeled with antibodies specific for actin, tubulin and other cytoskeletal components will be used to identify what cellular structures are responsible for the mechanical features seen in the AFM images. In addition, pharmacological agents will be used to disrupt specific structures, in particular cytoskeletal structures, and correlate that to loss of mechanical features. The successful completion of this project provides the necessary technology and scientific foundation for a complete study of cortical mechanics of the vascular endothelium, and mechanical remodeling in normal and diseased cells.