INHIBITION OF AIRWAY SMOOTH MUSCLE CONTRACTION

平滑肌收缩在急性气流阻塞中的作用 在哮喘中已经牢牢确立了。这背后的基本前提是 项目是长期抑制血管中的平滑肌收缩 哮喘可能为抗炎治疗提供有用的辅助手段 治疗这种流行和病态疾病的疗法。我们的主要目标是 创造一种新的策略，基于人造基因的表达， 抑制呼吸道的平滑肌收缩。我们已经克隆了 鉴定了编码SM22pha的全长小鼠基因，全长为22kd 在成年动物中仅表达于平滑肌肉的蛋白质。 我们计划的方法是使用SM22Alpha启动子来指导Smooth 抑制力量产生的多肽的肌肉特异性表达 肌动球蛋白。为了实现这些总体目标，我们提出了三点建议 具体目标：(1)确定限制顺式作用序列 SM22α在体内血管内皮细胞的表达我们之前 证实了小鼠SM22pha基因的BP-441到+41是 充分和必要地引导高水平的平滑肌细胞特异性 荧光素酶报告基因的体外表达。我们现在将确定 该基因在体内转录活性的分布 SM22pha启动子或更大的SM22pha启动子片段，在转基因中 这些调控元件控制LacZ基因表达的小鼠 记者基恩。这些研究将揭示一种特定于平滑肌的 将用于(下图)控制的转录调控元件 建议的治疗性基因在体内的表达。我们还将 确定过敏性呼吸道炎症的诱导是否改变 SM22Alpha启动子活性的大小或细胞分布 肺部。(2)建立SH-1肽MIRICRKK的能力 给药时抑制呼吸道平滑肌细胞的作用力 外源性或内源性合成血管内皮细胞 体外培养。SH-1多肽与骨骼肌肌动蛋白结合，抑制肌动蛋白-1 激活肌球蛋白ATPase活性，抑制皮肤作用力 骨骼肌纤维。我们将测试SH-1肽MIRICRKK 抑制去皮绵羊气管平滑肌肌动球蛋白力的产生 体外肌肉剥离，然后确认八肽已合成 用培养的气管平滑肌细胞转染人造影剂 编码MIRICRKK的微型基因。我们将测试MIRICRKK表达 在单个培养的牛气管肌细胞内抑制其 对缓激肽的收缩反应(使用原子力显微镜 测量横向细胞刚度的变化)，以及MIRICRKK 表达改变了心肌细胞的活性或增殖能力。这些 研究应该确定内源性合成的MIRICRKK 抑制肌动球蛋白力量的产生，以及它是否表现出毒性 效果。(3)评估呼吸道平滑肌感染是否与 携带人工基因的复制缺陷型腺病毒，其中 SM22pha启动子调控MIRICRKK的表达，抑制力量 产生对收缩激动剂的反应。我们将量化MIRICRKK 在绵羊、猫或人的呼吸道肌条中的表达 在体外感染，还是在体内感染猫气管虫，会和 这些组织与对照病毒的收缩反应- 感染或未感染的组织。我们预期这些研究将会 建立一种抑制呼吸道平滑肌收缩的新策略 得了哮喘。如果成功，这种方法可能会在其他 以血管或胃肠道平滑肌为特征的疾病 痉挛。

The role of smooth muscle contraction in causing acute airflow obstruction in asthma is firmly established. The basic premise underlying this project is that long-term inhibition of smooth muscle contraction in asthma may provide a useful adjunct to antiinflammatory therapy in the treatment of this prevalent and morbid disease. Our major goal is to create a new strategy, based on expression of an artificial gene, to inhibit smooth muscle contraction in the airways. We have cloned and characterized the full-length murine gene encoding SM22alpha, a 22 kd protein whose expression is restricted to smooth muscle in adult animals. Our planned approach is to employ the SM22alpha promoter to direct smooth muscle-specific expression of peptides that suppress force generation by actomyosin. To achieve these overall objectives, we propose three specific aims: (1) Identify the cis-acting sequences that restrict expression of SM22alpha to smooth muscle in vivo. We previously demonstrated that bp - 441 to +41 of the mouse SM22alpha gene are necessary and sufficient to direct high level smooth muscle cell-specific expression of a luciferase reporter gene in vitro. We will now identify in vivo the distribution of transcriptional activity of this minimal SM22alpha promoter or larger SM22alpha promoter fragments, in transgenic mice in which these regulatory elements control expression of the lacZ reporter gene. These studies will disclose a smooth muscle-specific transcriptional regulatory element that will be used (below) to control expression of the proposed therapeutic gene in vivo. We will also determine whether induction of allergic airway inflammation alters the magnitude or cellular distribution of SM22alpha promoter activity within the lung. (2) Establish the ability of the SH-1 peptide MIRICRKK to inhibit force generation by airway smooth muscle cells when delivered exogenously or when synthesized endogenously within smooth muscle cells in vitro. SH-1 peptides bind to skeletal muscle actin, inhibit actin- activated myosin ATPase activity, and inhibit force generation by skinned skeletal muscle fibers. We will test whether SH-1 peptide MIRICRKK inhibits actomyosin force generation in skinned sheep tracheal smooth muscle strips in vitro, then confirm that the octapeptide is synthesized by cultured tracheal smooth muscle cells transfected with an artificial minigene encoding MIRICRKK. We will test whether MIRICRKK expression within individual cultured bovine tracheal myocytes suppresses their contractile response to bradykinin (using atomic force microscopy to measure transverse cell stiffness changes), and whether MIRICRKK expression alters myocyte viability or proliferative capacity. These studies should establish whether endogenously synthesized MIRICRKK inhibits actomyosin force generation, and whether it exhibits toxic effects. (3) Evaluate whether infection of airway smooth muscle with replication-deficient adenovirus carrying an artificial gene, in which the SM22alpha promoter control expression of MIRICRKK, inhibits force generation in response to contractile agonists. We will quantify MIRICRKK expression within sheep, cat, or human airway smooth muscle strips infected in vitro, or cat trachealis infected in vivo, and will compare contractile responses of these tissues with those of control virus- infected or non-infected tissues. We anticipate that these studies will establish a novel strategy for inhibiting airway smooth muscle contraction in asthma. If successful, this approach might hold promise in other diseases characterized by vascular or gastrointestinal smooth muscle spasm.