Extracellular determinants of airway smooth muscle force: A new paradigm for sust

描述(申请人提供)：哮喘是一种慢性炎症性疾病，影响着美国2400万人和全球超过3亿人，而且其患病率还在上升。到目前为止，人们已经很好地认识到，慢性炎症会导致气道机械僵硬和组成的改变，称为“气道重塑”。此外，气道僵硬的变化意味着，在潮气呼吸期间，气道平滑肌(ASM)细胞将受到一种改变的张力模式的影响。这一提议解决了一个关键问题：ASM细胞细胞外环境机械结构的变化能否促使ASM细胞进入改变的基线状态，以便在暴露于激动剂时，它可以产生并维持能够放大呼吸道收缩的力量？这项提议试图回答这个问题并确定潜在的机制。在ASM中，肌动蛋白和肌球蛋白的收缩机制不仅以平行排列的收缩元素的形式存在，而且还以非常致密的肌动蛋白层与非肌肉亚型的肌球蛋白结合在细胞膜上，称为皮质肌动蛋白(CA)网络。我们的中心假设是，细胞外基质(ECM)硬度的变化可以导致CA网络主动重组为独特的构型，能够支持ASM夸大的力产生。这一假设源于我们开发的一个计算模型，该模型表明ASM细胞长时间维持力量的能力可以通过平行排列的肌球蛋白纤维与CA网络之间的机械相互作用来解释。同样的模型预测，当接触激动剂时，存在一定范围的ECM僵硬可以导致ASM产生非常高的力。为了从实验上验证这些想法，在这项提案的指导阶段，我将接受原代人类ASM细胞培养、细胞牵引力测量、肌动蛋白和其他细胞骨架组件的GFP标记以及活细胞成像的显微技术方面的培训。这将被用来实验确定ECM刚度对ASM可以产生的基线和主动力的影响，并确定CA网络结构和ASM力之间的联系。在此之后，我希望转型为一名独立的调查员。在R00阶段，我将研究拉伸和深呼吸对皮质肌动蛋白网络几何形状的影响，以及ASM细胞如何使用外部施加的应变被驱动到高或低力产生区域，最后在目标3中，我将确定这些结果如何扩展到ASM细胞的多细胞集合，重点是了解拉伸和细胞外基质硬度对ASM细胞之间耦合强度的影响。最终，这项研究可能会得出一种新的基于细胞和分子的范式，解释嵌入重塑的气道中的ASM如何以及为什么会变得过度反应，以及动态力在维持或消融这种情况中所起的作用。

DESCRIPTION (provided by applicant): Asthma is a chronic inflammatory disease that affects over 24 million people in the US and over 300 million people worldwide and its prevalence is rising. It is well recognized by now that chronic inflammation leads to an altered mechanical stiffness and composition of the airways referred to "airway remodeling". Further, changes in airway stiffness implies that, during tidal breathing, airway smooth muscle (ASM) cells will be subjected to an altered pattern of strains. This proposal addresses a critical question: Can the alterations in the mechanics of its extracellular environment drive the ASM cells into an altered baseline state such that upon agonist exposure, it can generate and sustain a force capable of amplified airway constriction? This proposal seeks to answer this question and identify the underlying mechanisms. Within the ASM, the contractile machinery of actin and myosin exists not only in the form of parallel arrays of contractile elements, but also as a very dense layer of actin close to cell membrane with non-muscle isoforms of myosin, referred to as the cortical actin (CA) network. Our central hypothesis is that alterations in the stiffness of the extracellula matrix (ECM) can cause the CA network to actively reorganize into unique configurations capable of sustaining exaggerated force generation by the ASM. This hypothesis derives from a computational model, that we developed, which shows that the ability of the ASM cells to maintain force for long periods of time can be explained by mechanical interactions between parallel array of actin myosin fibers and the CA network. The same model predicts that there are certain regimes of ECM stiffness that can cause the ASM generate very high forces when exposed to agonist. To experimentally verify these ideas, during the mentored phase of this proposal, I will gain training in primary human ASM cell culture, cellular traction force measurement, GFP labeling of actin and other cytoskeletal components and microscopy techniques for live cell imaging. This will be used to experimentally establish the effect of ECM stiffness on baseline and active force that an ASM can generate and determine a link between CA network structure and ASM force. Following this, I hope to transition into an independent investigator. During the R00 phase, I will examine the effect of stretch and deep breathing on cortical actin network geometry and how ASM cells can be driven into high or low force generating regimes using externally imposed strains and finally in aim 3, I will determine how these results scale to a multi-cellular ensemble of ASM cells with specific focus on understanding the effect of stretch and ECM stiffness on the strength of the coupling between ASM cells. Ultimately, this research may derive a new cellular and molecular based paradigm that explains how and why the ASM embedded in a remodeled airway can become hyperreactive and the role that dynamic forces play in sustaining or ablating this condition.