FACTORS DETERMINING HYPERREPSONSIVENESS FOR INTACT AIRWAYS

描述(由申请人提供)：气道平滑肌(ASM)在哮喘的气道高反应性(AHR)表型中起什么作用？大量研究证实，分离的ASM的长度振荡可以调节和减轻其对激动剂的净反应。在分子水平上出现了两个相关的假说来解释单独的ASM发现，即对拉伸的正常反应来自于ASM细胞的肌动蛋白-肌球蛋白交叉桥和/或细胞骨架流态化的扰乱平衡。然而，对于一个完整的气道，从ASM研究到实际AHR的分子水平假说之间存在差距，因为它在三维上收缩。许多重要的相互作用发生在一个完整的气道的细胞外基质(ECM)内，它可以影响ASM的收缩能力，从而影响气道的在位收缩。我们已经开发了一种独特的基于超声成像的系统来动态探测完整的呼吸道。这里，ASM处于其自然几何状态，嵌入在气道壁的ECM中，而气道壁暴露于生理相关的跨壁压力(PTM)波动。该系统允许在任何生理的PTM波动和/或诱发的收缩期间，在完整的气道的全长上同时实时测量管腔直径和壁厚。这些测量使我们能够计算完整的呼吸道系统的一组广泛的宏观机械特性。我们还可以应用生化和组织学方法来检查ASM细胞和ECM纤维的微观特性。这些初步数据表明，在完整的呼吸道中，PTM的变异可能会激活跨桥脱离和/或肌动蛋白解聚的细胞机制(可能与细胞骨架流态化有关)，但不一定会导致呼吸道扩张。基于此，我们建议检验以下假设：假设：在完整的气道系统中，生理呼吸过程中跨壁压力的变化不足以减弱反应性，因为气道壁ECM的机械特性阻止了ASM跨桥循环和肌动蛋白聚合的有效破坏。推论：在体内，哮喘的AHR不能简单地解释为无法适当地使ASM应变。目的1：探讨动态PTM变化对完整呼吸道反应性的影响。目的2：确定动态PTM变化对完整呼吸道的细胞内和细胞外影响。目的3：确定气道壁结构成分和ASM细胞过程如何影响暴露于动态跨壁压力变化的完整气道的反应性。这项建议将解决关键问题，即与ASM收缩相关的机制是否以及如何与动态和复杂的完整呼吸道系统相关，从而与调节气道反应性相关。我们的建议代表了理解与呼吸道高反应性相关的机制的重要一步。

DESCRIPTION (provided by applicant): What role does the airway smooth muscle (ASM) play in producing the asthmatic phenotype of airway hyperresponsiveness (AHR)? A plethora of studies confirm that length oscillations of isolated ASM can modulate and mitigate its net response to an agonist. Two related hypotheses at the molecular level have emerged to explain isolated ASM findings, namely that the normal responses to stretch arise from perturbed equilibrium of actin-myosin crossbridges and/or cytoskeletal fluidization of the ASM cell. However, a gap exists in bridging molecular level hypotheses from isolated ASM studies to actual AHR for an intact airway as it constricts in three-dimensions. Many important interactions occur within an intact airway's extracellular matrix (ECM) that can impact ASM contractility and hence airway constriction in situ. We have developed a unique ultrasound imaging-based system to dynamically probe intact airways. Here, the ASM is in its natural geometric state embedded within the airway wall's ECM, and the airway is exposed to physiologically relevant transmural pressure (Ptm) fluctuations. This system allows for concurrent real-time measurements of luminal diameter and wall thickness over the full length of an intact airway during any physiological Ptm fluctuations and/or induced constriction. These measurements allow us to calculate an extensive set of macroscopic mechanical properties of the intact airway system. We can also apply biochemical and histological approaches to examine the microscopic properties of the ASM cells and ECM fibers. Jointly, these preliminary data suggest in intact airways, Ptm variations may invoke cellular mechanisms of crossbridge detachment and/or actin de-polymerization (perhaps associated with cytoskeletal fluidization) but without necessarily resulting in airway dilation. Based on this, we propose to test the following hypothesis: HYPOTHESIS: In the intact airway system, transmural pressure variations during physiological breathing are insufficient to attenuate responsiveness because the mechanical properties of the airway wall's ECM prevent the effective disruption of ASM crossbridge cycling and actin polymerization. Corollary: In vivo, AHR in asthma cannot be explained simply as the inability to properly strain the ASM. Aim 1: To determine the contribution of dynamic Ptm variations to the responsiveness of intact airways. Aim 2: To determine the intra- and extracellular consequences of dynamic Ptm variations on intact airways. Aim 3: To determine how airway wall structural constituents and ASM cellular processes impact the responsiveness of intact airways exposed to dynamic transmural pressure variations. This proposal will address the crucial questions of if and how mechanisms associated with ASM contraction in isolation are relevant in a dynamic and complex intact airway system and, hence, relevant in modulating airway responsiveness. Our proposal represents an essential step to understand mechanisms relevant to airway hyperresponsiveness.