Mechanics and Molecular Mobility of Endothelial Cells

内皮细胞的力学和分子迁移率

• 批准号: 6922473
• 负责人: PETER J BUTLER
• 金额: $ 30.39万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6922473
• 关键词: biological transport; biomechanics; cell membrane; cell motility; confocal scanning microscopy; fluid flow; fluorescence microscopy; fluorescence polarization; fluorescence spectrometry; lipid transport; mathematical model; membrane activity; membrane lipids; nonblood rheology; physiology; shear stress; tissue /cell culture; vascular endothelium; viscosity

DESCRIPTION (provided by applicant): Blood flow-related shear stress induces biochemical and physiological changes in vascular endothelial cells (ECs) through membrane-mediated mechanisms. To understand the molecular basis of plasma membrane-mediated mechanotransduction, we propose new engineering analyses and experimental studies of single EC mechanotransduction. Central to our approach is the novel use of multimodal microscopy including DIG, TIRFM, confocal fluorescence imaging, time-resolved fluorescence, and photonic-force microscopy, all on a single platform. This infrastructure provides experimentally-determined inputs to advanced 3-D image processing algorithms, computational fluid dynamics solvers, and finite element (FE) solid mechanics models enabling time-and position-dependent correlations of cell membrane stresses with lipid-mediated signal transduction. To test our hypothesis that shear stress causes membrane stresses which elicit G-protein activation in gel-phase lipid microdomains we propose 3 specific aims (SAs). Under SA 1 we measure 3-D membrane topology, glycocalyx transport, and anisotropic membrane and cytoplasmic viscoelasticity to develop a full 3-D finite element mechanical model of an EC which predicts the shear-induced membrane stress distribution in the apical surface, cell junctions and focal adhesions. Under SA 2 we test the hypothesis that membrane stress concentrations are correlated with measured shear-induced changes in gel-phase lipid mobility and G-protein activation in EC membranes using time-resolved fluorescence spectroscopy of membrane phase-specific lipoid dyes and BODIPY-GTP, a novel fluorescent ligand for activated-G-proteins. Under SA 3 we use a novel continuous flow waveform generator to test whether prevailing shear stress elicits adaptive changes in cytoplasmic and membrane microrheology and membrane signaling. Results will point to new molecular level interventions for vascular dysfunction and provide the basis for intelligent development of novel biomaterials and tissue engineered blood vessels.

描述(申请人提供)：血流相关的剪应力通过膜介导的机制诱导血管内皮细胞(ECs)发生生化和生理变化。为了了解质膜介导的机械转导的分子基础，我们对单个EC的机械转导提出了新的工程分析和实验研究。我们方法的中心是多模式显微镜的新使用，包括地高辛、TIRFM、共聚焦荧光成像、时间分辨荧光和光子力显微镜，所有这些都在一个平台上。这一基础设施为先进的3-D图像处理算法、计算流体动力学解算器和有限元(FE)固体力学模型提供了实验确定的输入，从而实现了细胞膜应力与脂质介导的信号转导的时间和位置相关。为了验证我们的假设，即剪切力引起膜压力，从而诱导凝胶相脂微区中G蛋白的激活，我们提出了3个特定的目标(SA)。在SA-1下，我们测量了三维膜的拓扑结构、糖基化转运、各向异性膜和细胞质的粘弹性，以建立EC的全三维有限元力学模型，该模型预测了剪切诱导的膜应力在根尖表面、细胞连接和灶性粘连中的分布。在SA 2下，我们使用膜相特异的类脂染料的时间分辨荧光光谱和一种新的激活G蛋白的荧光配体BODIPY-GTP来验证这一假设，即膜压力浓度与测量到的剪切诱导的EC膜凝胶相脂流动性和G蛋白激活的变化有关。在SA3下，我们使用一种新型的连续流动波形发生器来测试普遍的剪切应力是否引起细胞质和膜的微观流变学和膜信号的适应性变化。研究结果将为治疗血管功能障碍提供新的分子水平的干预措施，并为新型生物材料和组织工程血管的智能开发提供基础。