Mechanical Feedback on Cell Structure and Signaling

描述（由申请人提供）：血管内皮细胞（EC）由于脉动压力而不断受到周期性拉伸。在直的、无分支的动脉中，EC是细长的，其主轴与动脉血管的主轴定向，即垂直于拉伸方向。垂直于拉伸方向排列的纤维比平行于拉伸方向排列的纤维承受更小的张力。因此，应力纤维的垂直取向用于降低拉伸诱导的张力，从而使单轴拉伸产生的细胞内机械能最小化。我们提出了一个机械和分子反馈控制模型，导致能源最小化EC应力纤维重塑。我们的初步实验表明，在EC中Rho活性的增加增强了应力纤维的垂直取向拉伸和Rho活性的抑制导致应力纤维的取向平行于拉伸的方向。这些结果使我们提出，Rho GTdR通过控制应力纤维重组在调节细胞内机械能的拉伸产生中具有核心作用。我们建议用以下特定目标下列出的研究来测试我们的模型：（1）为了确定在机械拉伸期间从测量的细胞粘弹性和应变率的细胞内能量的变化：磁扭转细胞术和细胞内颗粒位移将用于确定细胞粘弹性，其与拉伸诱导的应变率的测量一起，将允许计算细胞内能量。(2)为了确定不同模式的机械拉伸对应力纤维方向和细胞内能量的影响：我们将测量由各种模式的拉伸产生的细胞内能量，并跟踪由于应力纤维重塑和肌球蛋白轻链活性调节引起的细胞内能量变化。(3)为了阐明Rho GT3通路在牵张诱导的应力纤维取向和细胞内能量变化中的作用：Rho/ROCK/MLC通路激活的时间过程以及调节其活性的效果将与应力纤维重塑和细胞内能量相关。(4)为了确定不同模式的拉伸对机械转导和EC增殖/凋亡的影响：将测试拉伸诱导的细胞内能量对MAPK活化的相关性及其在细胞命运中的作用。(5)为了测试离体动脉系统中体外结果的有效性：将在切除动脉的完整内皮中测量不同拉伸模式对应力纤维重塑、细胞内信号传导和细胞命运的影响。从这些拟议的研究中产生的结果将提供深入了解细胞的适应机制和生物功能的稳态响应拉伸。

DESCRIPTION (provided by applicant): Vascular endothelial cells (ECs) are constantly subjected to cyclic stretch due to the pulsatile pressure. In straight, unbranched arteries, ECs are elongated with their major axes oriented with that of the arterial vessel, i.e. perpendicular to the direction of stretch. Fibers aligned perpendicular to stretch bear less tension than when they are aligned parallel to stretch. Thus, perpendicular orientation of the stress fibers serves to reduce the stretch- induced tension and thus minimize the intracellular mechanical energy produced by uniaxial stretch. We propose a mechanical and molecular feedback control model that leads to energy minimization by EC stress fiber remodeling. Our preliminary experiments have shown that an increase of Rho activity in ECs enhances the perpendicular orientation of stress fibers to stretch and that the inhibition of Rho activity causes the stress fibers to be oriented parallel to the direction of stretch. These results led us to propose that Rho GTPase has a central role in regulating the stretch-generation of intracellular mechanical energy through the control of stress fiber re-organization. We propose to test our model with studies listed under the following specific aims: (1) To determine the change in intracellular energy from measured cell viscoelastic properties and strain rate during mechanical stretch: Magnetic twisting cytometry and intracellular particle displacement will be used to determine cell viscoelastic properties which, together with the measurement of stretch-induced strain rate, will allow the computation of intracellular energy. (2) To establish the effects of different modes of mechanical stretch on stress fiber orientation and intracellular energy: We will measure the intracellular energy resulting from various modes of stretch and track changes in intracellular energy due to stress fiber remodeling and modulation of myosin light chain activity. (3) To elucidate the role of Rho GTPase pathway in stretch-induced changes in stress fiber orientation and intracellular energy: The time courses of activation of the Rho/ROCK/MLC pathway, as well as the effects of modulating their activities, will be related to stress fiber remodeling and intracellular energy. (4) To determine the effects of different modes of stretch on mechanotransduction and EC proliferation/apoptosis: The relevance of stretch-induced intracellular energy on the activation of MAPKs and their role in cell fate will be tested. (5) To test the validity of the in vitro results in an ex vivo artery system: The effects of different modes of stretch on stress fiber remodeling, intracellular signaling and cell fate will be measured in the intact endothelium of excised arteries. The results generated from these proposed studies will provide insight into the cellular adaptation mechanisms and homeostasis of biological functions in responses to stretch.