INHIBITION OF AIRWAY SMOOTH MUSCLE CONTRACTION

抑制气道平滑肌收缩

• 批准号: 6355589
• 负责人: Julian Solway
• 金额: $ 28.77万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355589
• 关键词: actins; asthma; atomic force microscopy; bronchomotion; cats; gene therapy; genetic regulatory element; genetically modified animals; human tissue; laboratory mouse; muscle contraction; muscle relaxing factor; muscle strength; myosins; oligopeptides; peptide hormone biosynthesis; reporter genes; smooth muscle; tissue /cell culture; trachea

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.

平滑肌收缩在引起急性气流阻塞中的作用 在哮喘中已牢固确立。 这背后的基本前提 该项目的目的是长期抑制平滑肌收缩 哮喘可能为抗炎治疗提供有用的辅助手段 治疗这种流行病态的疾病。 我们的主要目标是 创建一个基于人工基因表达的新策略 抑制气道平滑肌收缩。 我们已经克隆并 表征了编码 SM22alpha（22 kd）的全长鼠基因 其表达仅限于成年动物平滑肌的蛋白质。 我们计划的方法是利用 SM22alpha 启动子来直接平滑 抑制力产生的肽的肌肉特异性表达 肌动球蛋白。 为实现这些总体目标，我们提出三点建议 具体目标：（1）识别限制的顺式作用序列 SM22α在体内表达至平滑肌。 我们之前 证明小鼠 SM22alpha 基因的 bp - 441 至 +41 指导高水平平滑肌细胞特异性的必要和充分 荧光素酶报告基因的体外表达。 我们现在将识别 体内这个最小转录活性的分布 SM22α启动子或更大的SM22α启动子片段，转基因 这些调节元件控制 lacZ 表达的小鼠 报告基因。 这些研究将揭示平滑肌特异性 将用于（如下）控制的转录调控元件 所提出的治疗基因在体内的表达。 我们还将 确定过敏性气道炎症的诱导是否会改变 SM22α启动子活性的大小或细胞分布 肺。 (2) 建立SH-1肽MIRICRKK的能力 分娩时抑制气道平滑肌细胞产生力 外源性或当平滑肌细胞内源性合成时 体外。 SH-1 肽与骨骼肌肌动蛋白结合，抑制肌动蛋白- 激活肌球蛋白ATP酶活性，抑制皮肤产生力 骨骼肌纤维。 我们将测试SH-1肽MIRICRKK是否 抑制皮羊气管平滑肌肌动球蛋白力的产生 体外肌肉条，然后确认八肽合成 由培养的气管平滑肌细胞转染人工 编码 MIRICRKK 的小基因。 我们将测试 MIRICRKK 是否表达 在个体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 测量横向细胞刚度变化），以及是否 MIRICRKK 表达改变心肌细胞活力或增殖能力。 这些 研究应确定是否内源合成 MIRICRKK 抑制肌动球蛋白力的产生，以及是否表现出毒性 影响。 (3)评估气道平滑肌是否感染 复制缺陷型腺病毒携带人工基因，其中 SM22α 启动子控制 MIRICRKK 的表达，抑制力 响应收缩激动剂的产生。 我们将量化 MIRICRKK 在绵羊、猫或人气道平滑肌条中表达 体外感染，或体内猫气管感染，并将进行比较 这些组织与对照病毒的收缩反应 感染或未感染的组织。 我们预计这些研究将 建立抑制气道平滑肌收缩的新策略 在哮喘中。 如果成功的话，这种方法可能会在其他方面带来希望 以血管或胃肠道平滑肌为特征的疾病 痉挛。