Pulmonary arterial endothelial stiffness and shear-stress induced signaling

 描述(由申请人提供)：肺动脉高压(PAH)是一种破坏性的疾病。肺动脉(PA)僵硬的增加是PH患者死亡率的一个强有力的预测因子。在血流过程中，切应力诱导的与一氧化氮(NO)、钙(Ca~(2+))和三磷酸腺苷(ATP)有关的内皮细胞(EC)信号在血管平滑肌细胞僵硬中发挥重要作用，从而导致动脉僵硬。内皮细胞的局部剪切硬度如何影响ATP的产生、释放、NO和钙信号转导尚不清楚，因为在模拟血流过程中还不能测量内皮细胞的局部硬度。由于细胞僵硬与皮质下肌动蛋白张力有关，我们的假设S认为，在肺动脉高压中，单个肺动脉内皮细胞僵硬增加，作为局部血管活性介质信号的种子，通过皮质下肌动蛋白和微管直接机械转导到线粒体，从而减少ATP的产生和释放。我们建议通过使用正常和GATA-6缺失的PAH小鼠的内皮细胞来验证这一假设：目的1：建立一种体外方法，使用特定的量子点来测量培养的内皮细胞在模拟血流过程中的剪切硬度。目的：将细胞内剪切力与细胞内钙、一氧化氮、线粒体三磷酸腺苷的生成量和三磷酸腺苷水平的局部分布相关联，以确定血管内皮细胞刚性、钙和血管活性物质之间的生物学联系。目的：通过选择性抑制皮质下肌动蛋白和微管，确定细胞机械成分在切应力诱导的NO、ATP和钙信号转导中的作用。我们已经建立了一个微流体室，在对细胞内结构进行成像的同时，将培养中的细胞暴露在受控剪应力下。我们还开发了一个新的概念，允许我们通过计算细胞沿线的剪应力和结合到细胞表面的荧光微珠的位移成像来计算单个细胞的剪切刚度。我们将使用量子点来测量纳米尺度的微珠位移，以估计单个细胞的硬度。我们已经成功地标记了各种细胞内结构的流动中的细胞。我们还观察了线粒体内膜电压在细胞培养的双轴拉伸过程中作为ATP产生的替代指标，并表明线粒体的机械转导严重依赖于皮质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，测试与细胞僵硬和信号传递相关的想法，而这些想法是不可能的。此外，如果我们的假设得到证实，我们将对PAH中与剪应力相关的EC信号的早期事件有更好的机制基础。如果这个为期两年的项目成功，我们将使用结果作为一个完整的R01申请的初步数据。我们将扩展该方法以可视化正常和高血压大鼠的PA组织条在生理脉动血流波形下的细胞内结构和细胞硬度，并测试剪应力诱导的EC信号、血管平滑肌收缩和长期血管壁重塑之间的相互作用。

 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.