Identifying Molecular Phenotype of Normal and Asthmatic Bronchial Smooth Muscle

描述(申请人提供)：哮喘是一种常见疾病，有几种有效的治疗方法，包括吸入类固醇和激动剂。尽管如此，一小部分患者有与呼吸道重塑相关的严重的无情病程。气道重塑的一个主要特征是支气管平滑肌(BSM)表型的改变，典型的特征是细胞数量和大小的扩大，以及对特定和非特异性激动剂的高反应性增加。虽然哮喘的发病机制及其临床表现是基本的，但对错乱的BSM表型的基础缺乏了解在该领域是一个持续的、尚未解决的问题。这表现在缺乏关于潜在的支气管高反应性的分子信号的信息，以及缺乏专门针对逆转哮喘BSM表型的治疗。造成这种状况的一个主要因素是缺乏支持从哮喘肺中高保真分离纯BSM细胞进行分析的工具/方法。为了克服这一问题，我们开发了一种独特的转基因小鼠，在该小鼠中，BSM单一表达绿色荧光蛋白(HrGFP)，而血管平滑肌表达绿色(HrGFP)和红色荧光蛋白(DsRed)；从而首次提供了一种可靠的方法，用于利用流式细胞术将这两个平滑肌细胞群中的每一个相互分离，并从其他肺细胞中分离出来。利用这只独特的小鼠，我们的计划是对BSM RNA进行全面的mRNA和miRNA分析，以检验以下广泛的假设：1)BSM表达明显的遗传特征，2)该特征与哮喘的BSM表型有关。我们的计划是使用图谱数据来生成正常和哮喘BSM中完整和差异表达的mRNAs和miRNAs的列表。通过生物信息学和功能研究，哮喘BSM中非调控的miRNAs、mRNA表达和活性信号通路的同一性之间的关系将被检验。在这项工作结束时，我们将启动一个进程来填补哮喘领域的一个明显的知识空白，并将为未来的各种研究奠定基础。 与公共卫生相关：哮喘涉及到支气管周围肌肉的功能和属性的变化。虽然与哮喘相关的许多症状(包括喘息和呼吸短促)是核心，但导致支气管肌肉变化的分子信号却鲜为人知。这项建议的目的是使用我们开发的几种独特的工具和方法来识别哮喘中支撑支气管平滑肌变化的关键分子信号。

DESCRIPTION (provided by applicant): Asthma is a common disease with several effective treatments, including inhaled steroids and ¿-agonists. Despite this, a small subpopulation of patients has a severe unrelenting course associated with airway remodeling. A central feature of airway remodeling is alteration in bronchial smooth muscle (BSM) phenotype, which is classically characterized by expansion of cell number and size, and increased hyper- reactivity to specific and non-specific agonists. While fundamental to asthma pathogenesis and its clinical manifestations, a lack of knowledge regarding the basis for a deranged BSM phenotype is an ongoing, unresolved, issue in the field. This is manifested by the paucity of information regarding the molecular signals underlying bronchial hyper-reactivity and by the lack of treatments directed specifically at reversing the asthmatic BSM phenotype. One major contributing factor to this state-of-affairs is the lack of tools/methodologies that support the high fidelity isolation of pure BSM cells from asthmatic lungs for analysis. To overcome this, we developed a unique transgenic mouse in which BSM singly express a green fluorescent protein (hrGFP) whereas vascular smooth muscle express green (hrGFP) and red fluorescent proteins (dsRed); thereby providing for the first time a reliable methodology for separating each of these two smooth muscle cell populations from one another, and from other lung cells using flow cytometry. Using this unique mouse, our plan is to perform comprehensive mRNA and miRNA profiling of BSM RNA to test the following broad based hypothesis: 1) BSM express a distinct genetic signature and 2) alterations in this signature mediate asthmatic BSM phenotypes. Our plan is to use the profiling data to generate lists of complete and differentially expressed mRNAs and miRNAs in normal and asthmatic BSM. Relationships between deregulated miRNAs, mRNA expression, and the identity of active signaling pathways in asthmatic BSM will be examined by bioinformatic and functional studies. At the end of this work, we will have initiated a process to fill a marked knowledge void in the asthma field and will have established a foundation for a variety of future studies. PUBLIC HEALTH RELEVANCE: Asthma involves changes in the function and properties of the muscle that surrounds the bronchial tube. While central to the many of the symptoms associated with asthma including wheezing and shortness of breath, the molecular signals that cause changes in bronchial muscle are poorly understood. The objective of this proposal is to use several unique tools and methodologies that we developed to identify key molecular signals that underlay the change in bronchial smooth muscle in asthma.