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Micromechanical Characterization of Endothelial Cortex

内皮皮质的微机械表征

基本信息

批准号：
6865488
负责人：
JAN H HOH
金额：
$ 16.35万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2007-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6865488
关键词：
atomic force microscopy bioimaging /biomedical imaging biomechanics cell component structure /function cell morphology confocal scanning microscopy cytoskeleton imaging /visualization /scanning immunologic assay /test vascular endothelium vascular endothelium permeability

项目摘要

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.

这项研究的长期目标是了解细胞力学和血管内皮屏障功能之间的关系。内皮细胞通透性的改变在血管生成、关节硬化和炎症等过程中是重要的。血管内皮细胞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，这样的细胞骨架重组的结果在局部机械性能的重塑，这反过来又会有重要的后果屏障功能。因此，我们提出了一个努力，以表征模型内皮细胞（牛肺动脉细胞）的皮质的局部机械结构，并研究这些细胞的机械重塑。为了实现这一目标，我们在这里提出了一个试点项目，以开发必要的可视化皮质力学成像方法，并建立一个明确的细胞骨架和皮质机械架构之间的联系。该试点项目有两个具体目标。首先，我们将使用原子力显微镜，以产生高分辨率的图像，在活内皮细胞的皮质局部机械结构。这些图像将揭示这些细胞的皮质是如何组织的，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表征局部力学，并将这些测量与皮质形态学相关联。第二，我们将确定的分子组成部分，决定血管内皮细胞的机械结构，通过使用生化标记和药理学的方法。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的共聚焦显微镜之间的相关成像将用于确定什么样的细胞结构负责AFM图像中看到的机械特征。此外，药理学试剂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相关联。该项目的成功完成为全面研究血管内皮的皮质力学以及正常和病变细胞的机械重塑提供了必要的技术和科学基础。