Role of Mitochondria in Airway Smooth Muscle

 描述(由申请人提供)：拟议的研究将解决有关炎症诱导的人类呼吸道平滑肌(HASM)过度活动(收缩)和增殖(合成)状态的潜在机制的基本问题，这些状态是哮喘的特征。确定这些机制是开发新的哮喘治疗靶点的关键。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Mfn2(Mfn2)表达减少，这一途径通过触发高反应(收缩)和增殖(合成)状态在哮喘中发挥核心作用。具体目标1：确定炎性细胞因子对SR/ER应激、Mfn2表达和线粒体碎裂的影响。为此，我们将使用正常和哮喘患者分离的hASM细胞和组织来验证这样的假设，即炎性细胞因子触发SR/ER应激至少部分是由于ROS产生的增加，以及SR/ER应激导致Mfn2表达减少和线粒体碎裂增加。特定目的2：确定SR/ER应激和Mfn2表达减少对功能的影响。为此，我们将使用分离的正常和哮喘患者的hASM细胞和组织来检验这一假设，即在hASM中，炎性细胞因子诱导SR/ER应激和Mfn2表达减少使线粒体和SR/ER解偶联，从而减少线粒体钙缓冲导致[Ca2+]Cyt升高，并迫使对激动剂刺激的反应。特定目的3：确定SR/ER应激和Mfn2减少对hASM细胞增殖的影响。为此，我们将使用分离的正常和哮喘患者的hASM细胞和组织来验证炎性细胞因子通过减少Mfn2的表达而促进hASM细胞增殖的假设。具体目标4：确定针对SR/ER应激的治疗方法在缓解哮喘小鼠模型的气道高反应性和重塑方面的有效性。为此，我们将使用混合变应原小鼠模型来测试以下假设：使用化学伴侣(如4-PBA、TUDCA)靶向SR/ER应激将提供一种有效的治疗策略，逆转与哮喘相关的气道过度活动和钝性重塑。

 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.