Molecular basis of age-dependent changes in airway smooth muscle functions

描述（由申请方提供）：临床研究表明，随着年龄的增长，呼吸功能会显著丧失，导致生活质量下降，其他疾病的倾向增加，以及气雾剂药物递送治疗阻塞性呼吸系统疾病无效。然而，这种年龄依赖性呼吸丧失的细胞和分子基础尚不清楚。气道平滑肌（ASM）的收缩状态通过影响气道直径而在调节呼吸中起重要作用，因此ASM是用于治疗阻塞性气道疾病的药物的靶点。在这里，我们建议建立分子变化，发生在ASM中，占呼吸功能丧失的老年人使用气道和ASM从大鼠和人类受试者。初步研究表明，与“年轻”和“早期老年”大鼠相比，老年（“老年”）大鼠中ASM的收缩和舒张反应减弱。额外的数据表明，老化促进ASM中的表型“转换”，其中平滑肌收缩表型改变为增殖/合成表型。与年轻大鼠相比，从老年大鼠获得的ASM中肌球蛋白重链和平滑肌α-肌动蛋白（收缩表型标志物蛋白）的表达较低。此外，ASM细胞的总体转录组分析揭示了肌生长抑制素的表达降低，肌生长抑制素是已知抑制肌细胞增殖的TGF-β家族的成员。最后，ASM细胞的初步数据表明，随着年龄的增长，细胞内信号传导减少到收缩剂和舒张剂，激活ASM上的G蛋白偶联受体（GPCR）。基于这些研究，我们假设ASM经历表型调制与年龄的结果，在收缩和舒张反应性降低。我们进一步假设ASM收缩的3个关键调控特征的改变是这种表型转换的基础：1）GPCR信号传导/药物-机械耦合; 2）机电耦合; 3）动态细胞骨架组装。最后，我们假设ASM肌肉生长抑制素的表达减少是随着年龄增长而驱动表型调节的主要上游机制。在具体目标1中，我们建议使用大鼠和人类气道以及来自三个不同年龄组的ASM细胞，使用新型工具如肌描记器，光磁扭转细胞术和牵引显微镜来建立ASM表型的变化。在具体目标2中，我们建议通过识别GPCR信号传导和药物机械耦合、膜去极化/超极化及其效应物的调节以及ASM细胞细胞骨架组装的调节中与年龄相关的变化，建立年龄导致ASM收缩/舒张能力丧失的机制基础。目的3将探讨肌生长抑制素作为ASM表型中年龄相关变化的主要上游驱动因素的机制作用，以及目的2中详述的收缩功能相关机制。总的来说，这些研究旨在确定ASM功能的年龄依赖性分子变化，这些变化导致老年人群中呼吸功能的年龄相关性下降。我们的发现可能有助于开发改善呼吸功能的工具，并修改老年阻塞性肺疾病的诊断和治疗方案。 公共卫生相关性：老年人群的呼吸下降导致呼吸困难和呼吸衰竭。气道平滑肌是调节呼吸的重要结构成分，因此是大多数抗哮喘药物的靶点。这项研究工作中提出的研究将确定气道平滑肌中的细胞和分子变化，这些变化是呼吸的年龄依赖性变化的原因。这些发现将有助于建立年龄依赖性呼吸丧失的机制基础，并理想地确定改善呼吸的策略，并修改诊断工具和治疗老年人阻塞性肺病的方案。

DESCRIPTION (provided by applicant): Clinical studies have demonstrated a significant loss of respiratory function with age that results in reduced quality of life, increased propensity for other diseases, and ineffective aerosol drug delivery for the treatment of obstructive respiratory diseases. However, the cellular and molecular basis for this age-dependent loss of respiration is unknown. Airway smooth muscle (ASM) contractile state plays a significant role in regulating respiration by influencing the airway diameter and hence ASM is the target of drugs used in the treatment of obstructive airway diseases. Herein we propose to establish molecular changes that occur in the ASM that account for loss of respiratory function in the elderly using airways and ASM obtained from rats and human subjects. Preliminary studies demonstrate diminished contractile and relaxation responses of ASM in aged ("Old") rats compared to "Young" and "Early Aged" rats. Additional data suggest aging promotes phenotype "switching" in ASM in which the smooth muscle contractile phenotype is changed to a proliferative/synthetic phenotype. Expression of myosin heavy chain and smooth muscle a-actin (contractile phenotype marker proteins) is lower in ASM obtained from Old rats compared to Young rats. Furthermore, global transcriptome analyses of ASM cells reveal decreased expression of myostatin, a member of the TGF-b family known to inhibit the proliferation of myocytes. Lastly, preliminary data from ASM cells suggest diminished intracellular signaling with age to both contractile and relaxant agents that activate G protein-coupled receptors (GPCRs) on ASM. Based on these studies we hypothesize that ASM undergoes phenotype modulation with age that results in decreased contractile and relaxant responsiveness. We further hypothesize that alterations in 3 key regulatory features of ASM contractile underlie this phenotype switch: 1) GPCR signaling/pharmaco-mechanical coupling; 2) electromechanical coupling; and 3) dynamic cytoskeleton assembly. Lastly, we hypothesize that reduced expression of ASM myostatin in ASM is the principal upstream mechanism driving phenotype modulation with age. In Specific Aim 1, we propose to establish changes in ASM phenotype using rat and human airways and ASM cells from three distinct age groups, using novel tools such as myograph, optical magnetic twisting cytometry and traction microscopy. In Specific Aim 2, we propose to establish the mechanistic basis by which age causes a loss of the contractile/relaxant capacity of ASM by identifying age-related changes in GPCR signaling and pharmaco-mechanical coupling, regulation of membrane de/hyper-polarization and its effectors, and in the regulation of ASM cell cytoskeleton assembly. Aim 3 will explore the mechanistic role of myostatin as the principal upstream driver of the age-related change in ASM phenotype and those mechanisms underlying contractile function detailed in Aim 2. Collectively, these studies seek to identify age-dependent molecular changes in ASM function that contribute to the age-associated decline in respiratory function in the elderly population. Our findings may help develop tools to improve respiratory functions, and modify diagnostic and treatment regimens for obstructive pulmonary diseases in the elderly. PUBLIC HEALTH RELEVANCE: Respiration declines in the elderly population leading to difficulty in breathing and respiratory failure. Smooth muscle lining the airways is an important structural component that regulates respiration and hence is a target for most anti-asthma medications. Studies proposed in this research work will identify cellular and molecular changes in the airway smooth muscle that account for age-dependent change in respiration. The findings will help establish a mechanistic basis for the age-dependent loss of respiration, and ideally identify strategies to improve respiration and modify diagnostic tools and regimens for treatment of obstructive lung diseases in the elderly.