Role of Mitochondria in Airway Smooth Muscle

 描述（由申请人提供）：拟定的研究将解决有关炎症诱导的人气道平滑肌（hASM）过度活跃（“收缩”）和增殖（“合成”）状态增强的机制的基本问题，这是哮喘的标志。识别这些机制是开发哮喘新治疗靶点的关键。我们的中心假设是，在hASM中，炎性细胞因子诱导肌内质网（SR/ER）应激，导致线粒体融合蛋白线粒体融合蛋白2（Mfn 2）的表达减少，并且该途径通过触发高反应性（收缩）和增殖性（合成）状态在哮喘中发挥核心作用。具体目的1：确定炎性细胞因子对SR/ER应激、Mfn 2表达和线粒体片段化的影响。在这个目标中，我们将使用解离的hASM细胞和组织从正常和哮喘患者来测试的假设，炎症细胞因子触发SR/ER应力至少部分由于增加ROS的产生，SR/ER应力导致Mfn 2的表达减少和线粒体片段化增加。具体目的2：确定SR/ER应激和Mfn 2表达降低的功能影响。在这个目标中，我们将使用正常和哮喘患者的解离hASM细胞和组织来检验以下假设：在hASM中，炎性丝氨酸诱导的SR/ER应激和Mfn 2表达减少使线粒体和SR/ER解偶联，从而减少线粒体Ca 2+缓冲，导致[Ca 2 +]cyt升高，并迫使对激动剂刺激的反应。具体目的3：确定SR/ER应激和减少的Mfn 2对hASM细胞增殖的影响。在这个目标中，我们将使用解离的hASM细胞和组织从正常和哮喘患者来测试的假设，即炎症细胞因子增加hASM细胞增殖的Mfn 2表达减少的结果。具体目标4：确定靶向SR/ER应激的治疗方法在缓解哮喘小鼠模型气道高反应性和重塑中的疗效。在这个目标中，我们将使用混合过敏原小鼠模型来测试使用化学分子伴侣（例如，4-PBA，TUDCA）将提供一种有效的治疗策略，以逆转与哮喘相关的气道过度活跃和钝性重塑。

 DESCRIPTION (provided by applicant): The proposed studies will address fundamental questions regarding the mechanisms underlying inflammation-induced enhancement of both hyperactive ("contractile") and proliferative ("synthetic") states of human airway smooth muscle (hASM), which are hallmarks of asthma. Identifying these mechanisms is the key to developing novel therapeutic targets for asthma. Our central hypothesis is that in hASM, inflammatory cytokines induce sarco-endoplasmic reticulum (SR/ER) stress leading to reduced expression of the mitochondrial fusion protein mitofusin 2 (Mfn2), and that this pathway plays a central role in asthma by triggering both hyper reactive (contractile) and proliferative (synthetic) states. Four Specific Aims are proposed: Specific Aim 1: To determine the impact of inflammatory cytokines on SR/ER stress, Mfn2 expression and mitochondrial fragmentation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines trigger SR/ER stress due at least in part to an increase in ROS generation, and that SR/ER stress leads to reduced Mfn2 expression and increased mitochondrial fragmentation. Specific Aim 2: To determine the functional impact of SR/ER stress and reduced Mfn2 expression. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that in hASM, inflammatory cytokine-induced SR/ER stress and reduced Mfn2 expression uncouples mitochondria and the SR/ER, thereby reducing mitochondrial Ca2+ buffering leading to elevated [Ca2+]cyt and force responses to agonist stimulation. Specific Aim 3: To determine the impact of SR/ER stress and reduced Mfn2 on hASM cell proliferation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines increase hASM cell proliferation as a result of decreased Mfn2 expression. Specific Aim 4: To determine the efficacy of therapeutic approaches targeting SR/ER stress in alleviating airway hyper reactivity and remodeling in a mouse model of asthma. In this aim, we will use a mixed allergen mouse model to test the hypothesis that targeting SR/ER stress using chemical chaperones (e.g., 4-PBA, TUDCA) will provide an effective therapeutic strategy to reverse airway hyperactivity and blunt remodeling associated with asthma.