Mechanics and Molecular Mobility of Endothelial Cells

描述（由申请方提供）：血流相关切应力通过膜介导机制诱导血管内皮细胞（EC）的生化和生理变化。为了了解质膜介导的机械力转导的分子基础，我们提出了新的工程分析和实验研究的单一EC机械力转导。我们的方法的核心是多模式显微镜的新用途，包括DIG，TIRFM，共聚焦荧光成像，时间分辨荧光和光子力显微镜，所有在一个平台上。该基础设施提供实验确定的输入，先进的3-D图像处理算法，计算流体动力学求解器，和有限元（FE）固体力学模型，使时间和位置依赖的相关性的细胞膜应力与脂质介导的信号转导。为了验证我们的假设，即剪切应力引起膜应力，引起G-蛋白激活凝胶相脂质微区，我们提出了3个具体的目标（SA）。根据SA 1，我们测量3-D膜拓扑结构，糖萼运输，各向异性膜和细胞质粘弹性开发一个完整的3-D有限元力学模型的EC预测的剪切诱导的膜应力分布在顶端表面，细胞连接和粘着斑。根据SA 2，我们测试的假设，膜应力浓度与测量的剪切诱导的凝胶相脂质流动性和G-蛋白激活EC膜的变化，使用时间分辨荧光光谱的膜相特异性类脂染料和BODIPY-GTP，一种新的荧光配体激活的G-蛋白。在SA 3下，我们使用一种新的连续流波形发生器来测试是否占主导地位的剪切应力elaborate细胞质和膜的微流变学和膜信号的适应性变化。研究结果将为血管功能障碍提供新的分子水平干预，并为新型生物材料和组织工程血管的智能开发提供基础。

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.