INHIBITION OF AIRWAY SMOOTH MUSCLE CONTRACTION

平滑肌收缩在急性气流阻塞中的作用 哮喘的发病率是很高的 这背后的基本前提是 长期抑制平滑肌收缩， 哮喘可能是哮喘治疗的一个有用的辅助手段， 治疗这种流行的病态疾病。 我们的主要目标是 创造一种新的策略，基于人工基因的表达， 抑制气道平滑肌收缩。 我们已经克隆和 其特征在于编码SM 22 α的全长鼠基因， 在成年动物中表达仅限于平滑肌的蛋白质。 我们计划的方法是采用SM 22 α启动子来直接平滑 肌肉特异性表达的肽，其抑制力产生， 肌动球蛋白 为了实现这些总体目标，我们提出三个 具体目标：（1）确定限制性的顺式作用序列 SM 22 α在体内平滑肌中的表达。 我们之前 表明小鼠SM 22 α基因的bp - 441至+41是 必要和足够的指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 在体内，这种最小的转录活性的分布 SM 22 α启动子或更大的SM 22 α启动子片段， 这些调控元件控制lacZ表达的小鼠 报告基因 这些研究将揭示一种平滑肌特异性 转录调控元件，其将用于（下文）控制 所提出的治疗基因的体内表达。 我们还将 确定过敏性气道炎症的诱导是否改变了 SM 22 α启动子活性的大小或细胞分布 肺 (2)建立SH-1肽MIRICRKK的能力， 当递送时抑制气道平滑肌细胞产生力 外源性或当在平滑肌细胞内内源性合成时， 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，并抑制力产生的皮肤 骨骼肌纤维 我们将测试SH-1肽MIRICRKK是否 抑制去皮绵羊气管平滑肌肌动球蛋白力的产生 在体外肌条中，然后确认八肽合成 通过用人工转染的培养的气管平滑肌细胞 编码MIRICRKK的小基因。 我们将测试MIRICRKK表达是否 在单个培养的牛气管肌细胞内抑制它们的 收缩反应缓激肽（使用原子力显微镜， 测量横向单元刚度变化），以及MIRICRKK 表达改变肌细胞活力或增殖能力。 这些 研究应该确定内源性合成的MIRICRKK 抑制肌动球蛋白力的产生，以及它是否表现出毒性 方面的影响. (3)评估是否感染气道平滑肌， 携带人工基因的复制缺陷型腺病毒，其中 SM 22 alpha启动子控制MIRICRKK的表达，抑制力 对收缩激动剂的反应。 我们将量化MIRICRKK 在绵羊、猫或人气道平滑肌条内表达 体外感染或猫气管感染，并将比较 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新策略 哮喘病 如果成功的话，这种方法可能会在其他领域带来希望。 以血管或胃肠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.