Nicotine, Airway Smooth Muscle and Asthma

摘要 虽然电子烟被宣传为哮喘患者吸烟的更安全的替代品，但越来越多的证据表明， 他们的肺毒性，需要更好地了解气道尼古丁生物学：临床相关的 未满足的需求和这项赠款的主要目标。在哮喘中，炎症增强气道平滑肌（ASM） Ca 2+（[Ca 2 +]cyt）和收缩性（气道高反应性; AHR），沿着涉及ASM的重塑 增殖考虑到吸入尼古丁可直接影响ASM，尼古丁对哮喘ASM的影响是 关键的，但鲜为人知的是烟碱受体（nAChR）的表达，其在人类ASM中的作用，或在哮喘。 人类ASM的初步数据显示功能性α7 nAChR：A）随着炎症和哮喘而增加， B）增强ASM [Ca 2 +]cyt/收缩性; D）激活促增殖途径。在混合过敏原的哮喘小鼠模型中观察到了这种效应 暴露于急性或慢性吸入尼古丁，但在α7抑制和α7 KO小鼠中减弱。所以我们 假设尼古丁通过ASM α7促进哮喘中的AHR和重塑。我们的目标是： 目的1：在人ASM中，确定α7在炎症和哮喘中的表达和调节机制。 目的2：在非哮喘患者与哮喘患者的人类ASM中，确定α7有助于 尼古丁增强AHR背景下的[Ca 2 +]cyt和收缩性。目的3：在非哮喘患者的人ASM中 vs.哮喘，以确定α7有助于尼古丁增强细胞增殖的机制。 目的4：在小鼠过敏性哮喘模型中，探讨α7在吸入尼古丁对AHR影响中的作用 和重塑。研究使用非哮喘患者与轻中度哮喘患者的成人ASM， 与ASM和哮喘相关的细胞因子（TNFα、IL-6、IL-13），伴/不伴尼古丁。目标1探索表达 和α7 nAChR，调节分子伴侣的定位，以及α7增加的机制， 炎症/哮喘（例如MAPK、PI 3/Akt、NFκB、Stats）。α7作为通道的功能使用 电生理学目的2探索急性与慢性尼古丁对[Ca 2 +]细胞调节（内流，SR Ca 2+）的影响 释放）和收缩性（牵引力、器官浴）。目的3探索尼古丁对ASM增殖的影响， ER应激、线粒体分裂（Drp 1，Fis 1）与融合（Mfn 1/2，Opa 1）和呼吸（3a）的作用，以及 精氨酸相关增殖途径（3b）。在这些目标中，α7的作用是使用宽与亚基- 特异性激动剂、拮抗剂或siRNA。体外结果整合在Aim 4中，使用成年小鼠模型， 哮喘伴/不伴急性与慢性吸入尼古丁。α7抑制剂（MG 624）的缓解作用和 检测平滑肌中的α7 KO。研究评估气道反应性、重塑和ASM生化 变化临床意义在于确定ASM α7作为减轻AHR和重塑的新靶点 哮喘和尼古丁一样。

ABSTRACT While e-cigarettes are advertised as safer alternatives to smoking for asthmatics, there is increasing evidence of their pulmonary toxicity, necessitating better understanding of airway nicotine biology: a clinically-relevant unmet need, and major goal of this grant. In asthma, inflammation enhances airway smooth muscle (ASM) Ca2+ ([Ca2+]cyt) and contractility (airway hyperreactivity; AHR), along with remodeling involving ASM proliferation. Given that inhaled nicotine can directly influence ASM, nicotine effects on asthmatic ASM are critical, but little is known about nicotinic receptor (nAChR) expression, its role in human ASM, or in asthma. Preliminary data in human ASM show functional α7 nAChRs that A) increase with inflammation and asthma, B) enhance ASM [Ca2+]cyt/contractility; C) enhance ER stress and mitochondrial fission, and respiration, and D) activate pro-proliferative pathways. Such effects are observed in a mixed-allergen mouse model of asthma exposed to acute or chronic inhaled nicotine, but blunted by α7 inhibition and in α7 KO mice. Thus, our hypothesis is that nicotine acts via ASM α7 to promote AHR and remodeling in asthma. Our Aims are: Aim 1: : In human ASM, determine mechanisms of α7 expression and regulation in inflammation and asthma. Aim 2: In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which α7 contributes to nicotine enhancement of [Ca2+]cyt and contractility in the context of AHR. Aim 3: In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which α7 contributes to nicotine enhancement of cell proliferation. Aim 4: In a mouse model of allergic asthma, to determine the role of α7 in effects of inhaled nicotine on AHR and remodeling. Studies use adult human ASM of non-asthmatics vs. mild-moderate asthmatics exposed to cytokines relevant to ASM and to asthma (TNFα, IL-6, IL-13), with/without nicotine. Aim 1 explores expression and localization of α7 nAChR, regulatory chaperones, and mechanisms by which α7 is increased in inflammation/asthma (e.g. MAPKs, PI3/Akt, NFκB, Stats). α7 functionality as a channel is tested using electrophysiology. Aim 2 explores acute vs. chronic nicotine effects on [Ca2+]cyt regulation (influx, SR Ca2+ release) and contractility (traction force, organ bath). Aim 3 explores nicotine effects on ASM proliferation, and the roles of ER stress, mitochondrial fission (Drp1, Fis1) vs. fusion (Mfn1/2, Opa1) and respiration (3a), and of cytokine-associated proliferative pathways (3b). In these Aims, role of α7 is determined using broad vs. subunit- specific agonists, antagonists or siRNAs. In vitro results are integrated in Aim 4 using the adult mouse model of asthma with/without acute vs. chronic inhaled nicotine. Alleviating effects of α7 inhibitor (MG624) and effects of α7 KO in smooth muscle are tested. Studies assess airway reactivity, remodeling, and ASM biochemical changes. Clinical significance lies in identifying ASM α7 as a novel target for alleviating AHR and remodeling of asthma as well as with nicotine.