Advanced Image-Based Approach to Assess How Fibrillar Collagen Modulates Airway Reactivity

 描述(由申请者提供)：传统上，哮喘的气道高反应性(AHR)的病因学研究的重点是气道平滑肌(ASM)。然而，越来越清楚的是，导致AHR的关键机制不能仅仅通过从其自然环境中孤立地了解ASM来收集。当ASM细胞收缩时，它们的力/变形必须围绕气道周围传播，以便使气道变窄。这通常被认为是通过ASM细胞之间的细胞-细胞接触发生的，每个ASM细胞拉动另一个ASM细胞试图缩小呼吸道。然而，除了细胞与细胞之间的相互作用外，ASM细胞还可以与ECM形成局灶性粘连。因此，ASM力可能通过环绕ASM细胞的细胞外基质成分(如纤维状胶原)在气道壁周围传递。从这一思路中产生的一个基本问题是：ASM对激动剂的反应如何转化为最终的气道狭窄，以及在狭窄过程中，胶原纤维在调节气道口径方面发挥了什么作用(如果有的话)。目前，这个问题是不可能解决的，因为对肌肉收缩期间气道壁中的胶原纤维发生了什么一无所知。我们的建议将推进一种创新和强大的方法，该方法将使用二次谐波产生(SHG)显微镜，一种新的成像技术，将胶原纤维组织与呼吸道内的应力传递联系起来，从而影响呼吸道的反应。我们的初步结果表明，SHG可以作为一种可行的技术来描绘由于ASM激活或循环拉伸而导致的胶原纤维组织的变化。此外，当我们在使用SHG实时成像的同时将新鲜解剖的气道段暴露在10-5M的乙酰胆碱(ACh)中时，我们发现与我们目前对ASM力如何在气道周围传递的理解相反，基线处的波状胶原纤维在ACH后图像中似乎随着气道的收缩而变直。这种伸直与当呼吸道接触激动剂和ASM产生活动力时，胶原纤维开始携带张力是一致的。如果得到证实，这意味着在ASM收缩过程中，气道壁胶原在传递气道力方面发挥了更直接的作用。通过这个探索性的研究方案，我们将为社区提供一个定量的了解，由ASM收缩或动态拉伸引起的胶原组织的变化，以及气道壁应力的变化，这些变化被映射到理解气道狭窄的过程。

 DESCRIPTION (provided by applicant): The airway smooth muscle (ASM) has traditionally been the focus of research into the etiology of airway hyperresponsiveness (AHR) in asthma. However, it is becoming increasingly clear that critical mechanisms leading to AHR cannot be gleaned by only understanding the ASM in isolation from its native environment. When the ASM cells constrict, their force/deformation has to propagate around the circumference of the airway in order for the airway to narrow. This is typically thought to occur through cell-cell contacts between ASM cells, with each ASM cell pulling on another ASM cell attempting to narrow the airway. However, in addition to the cell-cell interactions, it is well established that the ASM cels can form focal adhesions with the ECM. Therefore, it is possible that the ASM force can be transmitted around the airway wall through elements of the ECM such as fibrillar collagen that surround the ASM cells. A fundamental question that emerges from this line of thinking is: how does the ASM contraction in response to an agonist translate into the eventual narrowing of an airway and what role do the collagen fibers play, if any, in modulating airway caliber during the narrowing process. At present, it is impossible to address this question as nothing is known about what happens to the collagen fibers in the airway wall during a muscle contraction. Our proposal will advance an innovative and powerful approach that will use second harmonic generation (SHG) microscopy, a novel imaging technique, to link collagen fiber organization to stress transmission within the airway and consequently the response of airways. Our preliminary results indicate that SHG can be a viable technology to map out the changes in collagen fiber organization as a result of ASM activation or cyclic stretching. Further, when we exposed a freshly dissected airway segment to 10-5M acetylcholine (ACh) while imaging it real time using SHG, we found that counter intuitive to our present understanding of how an ASM force is transmitted around an airway, wavy collagen fibers at baseline appear to straighten in the post ACh image as the airway constricted. Such straightening is consistent with collagen fibers starting to carry tension when the airway is exposed to agonist and the ASM develops active force. If confirmed, this means that airway wall collagen plays a more direct role in transmitting force around the airway during an ASM contraction. Through this exploratory research proposal, we will provide the community with a quantitative understanding of changes in collagen organization caused by an ASM contraction or dynamic stretch and changes in airway wall stress that be mapped back to understand the process by which airways narrow.