Pulmonary arterial endothelial stiffness and shear-stress induced signaling

 描述（由申请人提供）：肺动脉高压（PAH）是一种破坏性疾病。肺动脉（PA）僵硬度的增加是PH患者死亡率的一个强有力的预测因素。在血流过程中，剪切应力诱导的内皮细胞（EC）信号传导与一氧化氮（NO），钙（Ca 2+）和ATP相关，似乎在血管平滑肌细胞僵硬度中发挥重要作用，有助于动脉僵硬度。很少有人知道如何局部剪切刚度的内皮层的影响ATP的生产，ATP的释放，NO和Ca信号，因为它一直没有可能来测量局部刚度的内皮细胞在模拟血流。由于细胞硬度与皮层下肌动蛋白张力有关，我们假设在肺动脉高压中，硬度增加的个体肺动脉EC作为局部血管活性介质信号传导的种子，通过皮层下肌动蛋白和微管直接机械转导到线粒体，减少ATP的产生和释放。我们建议使用来自正常和患有PAH的加塔-6无效小鼠的EC来测试这一假设，其目的如下：目的1：开发一种体外方法来测量在模拟血流期间使用特定量子点培养的EC的剪切刚度。目标二：将剪切刚度的地形分布与细胞内Ca 2+和NO、线粒体ATP产生和ATP水平相关联，以确定EC刚度、Ca 2+和血管活性介质之间的生物学联系。目标3：通过选择性抑制皮层下肌动蛋白和微管，确定细胞机械成分在剪切应力诱导的NO、ATP和Ca 2+信号传导中的作用。我们已经建立了一个微流体室，将培养中的细胞暴露于受控的剪切应力，同时对细胞内结构进行成像。我们还开发了一种新的概念，使我们能够计算剪切刚度的单个细胞通过计算剪切应力沿着细胞和成像的荧光珠结合到细胞表面的位移。我们将使用量子点来测量纳米级珠子位移，以估计单个细胞的刚度。我们已经成功地标记了各种细胞内结构的流动下的细胞。我们还可视化线粒体内膜电压作为ATP生产的替代品，在双轴拉伸培养的细胞，并表明线粒体mechanodonduction是严重依赖于皮层下肌动蛋白和微管组织。这些研究的意义在于，我们的新设备将开启新的潜力，以测试与细胞硬度和信号传导相关的想法，这是不可能的。此外，如果我们的假设得到证实，我们将有更好的机制基础的早期事件的剪切应力相关的EC信号在PAH。如果这个为期2年的项目是成功的，我们将使用的结果为一个完整的R 01应用的初步数据。我们将扩展的方法来可视化细胞内结构和测量细胞硬度PA组织条从正常和高血压大鼠生理脉动血流波形和测试剪切应力诱导的EC信号，血管平滑肌收缩和长期血管壁重塑的相互作用。

 DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a devastating disorder. An increase in pulmonary artery (PA) stiffness is a strong predictor of mortality in patients with PH. During blood flow, shear stress-induced endothelial cell (EC) signaling related to nitric oxide (NO), calcium (Ca2+) and ATP appears to play important roles in vascular smooth muscle cell stiffness that contributes to arterial stiffness. Little is known how local shear stiffness of the endothelial layer influences ATP production, ATP release, NO and Ca signaling because it has not been possible to measure the local stiffness of ECs during simulated blood flow. Since cell stiffness is related to subcortical actin tension, our hypothesis s that in pulmonary hypertension, individual pulmonary arterial ECs with increased stiffness act as seeds for local vasoactive mediator signaling which reduces ATP production and release through direct mechanotransduction to the mitochondria via the subcortical actin and microtubules. We propose to test this hypothesis with the following aims using ECs from normal and GATA-6 null mice with PAH: Aim 1: Develop an in vitro method to measure the shear stiffness of ECs in culture using specific quantum dots during simulated blood flow. Aim 2: Correlate the topographical distributions of shear stiffness with intracellular Ca2+ and NO, mitochondrial ATP production and ATP level, to identify biological links between EC stiffness, Ca2+ and vasoactive mediators. Aim 3: Determine the roles of cellular mechanical components in shear stress-induced NO, ATP and Ca2+ signaling through selective inhibition of subcortical actin and microtubules. We have built a microfluidic chamber to expose cells in culture to controlled shear stress while imaging intracellular structures. We have also developed a novel concept that allows us compute the shear stiffness of individual cells by computing shear stress along cells and imaging of displacements of fluorescent beads bound to cell surface. We will use quantum dots to measure nm-scale bead displacements to estimate the stiffness of individual cells. We have successfully labeled cells under flow for various intracellular structures. We have also visualized mitochondrial inner membrane voltage as a surrogate of ATP production, during biaxial stretch of cells in culture and showed that mitochondrial mechanotransduction is critically dependent on subcortical actin and microtubule organization. The significance of these studies is that our new device will open new potentials to test ideas related to cell stiffness and signaling that have not been possible. Additionally, if our hypothesi is confirmed, we will have much better mechanistic underpinnings of the early events in shear stress related EC signaling in PAH. If this 2-year project is successful, we will use the results a preliminary data for a full R01 application. We will extend the methodology to visualize intracellular structures and measure cell stiffness of PA tissue strips from normal and hypertensive rats under physiological pulsatile blood flow waveforms and test the interaction of shear stress-induced EC signaling, vascular smooth muscle contraction and longer-term vascular wall remodeling.