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

描述（由申请人提供）：哮喘是一种慢性炎症性疾病，在美国影响超过2400万人，全球超过3亿人，其患病率正在上升。众所周知，慢性炎症导致气道的机械刚度和组成改变，即“气道重塑”。此外，气道刚度的变化表明，在潮汐呼吸期间，气道平滑肌（ASM）细胞将受到应变模式的改变。这一建议解决了一个关键问题：细胞外环境机制的改变能否驱动ASM细胞进入改变的基线状态，从而在激动剂暴露后产生并维持一种能够放大气道收缩的力？本建议试图回答这个问题，并确定潜在的机制。在肌动蛋白中，肌动蛋白和肌凝蛋白的收缩机制不仅以平行排列的收缩元件的形式存在，而且还以非常致密的肌动蛋白层的形式与肌凝蛋白的非肌肉同型体靠近细胞膜，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）刚度的改变可以导致CA网络主动重组成独特的结构，能够维持ASM产生的夸张力。这一假设来源于我们开发的一个计算模型，该模型表明，肌动蛋白肌球蛋白纤维平行阵列和CA网络之间的机械相互作用可以解释肌动蛋白细胞长时间保持力的能力。同一模型预测，当暴露于激动剂时，ECM刚度的某些制度可能导致ASM产生非常高的力。为了在实验上验证这些想法，在本提案的指导阶段，我将接受人类原代ASM细胞培养，细胞牵引力测量，肌动蛋白和其他细胞骨架成分的GFP标记以及活细胞成像的显微镜技术的培训。这将用于实验建立ECM刚度对ASM可以产生的基线和主动力的影响，并确定CA网络结构与ASM力之间的联系。在此之后，我希望转变为一名独立调查员。在R00阶段，我将检查拉伸和深呼吸对皮质肌动蛋白网络几何形状的影响，以及如何使用外部施加的张力将ASM细胞驱动到高或低力产生机制，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，并特别关注拉伸和ECM刚度对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，来解释在重塑气道中嵌入的ASM如何以及为什么会变得高反应性，以及动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.