CIRCADIAN-COUPLED CELLULAR AND LUNG FUNCTION IN COPD

描述（由申请人提供）： 慢性阻塞性肺疾病（COPD）是美国慢性病发病率和死亡率的第四大原因。随着COPD进展，患者发生更频繁和严重的急性加重，急诊室就诊和住院率增加，特别是在夜间和白天早期，此时肺功能最低，类固醇和支气管扩张剂的影响最小。COPD患者存在异常的昼夜节律，反映在气道口径和阻力的昼夜变化以及呼吸道症状上。然而，COPD患者肺部昼夜节律改变的细胞和分子机制尚不清楚。生物钟周期蛋白（Per）和隐花色素（Cry）及其转录激活因子核心CLOCK和BMAL 1调节内在的每日节律，但这些分子振荡器在肺生理学和病理学中响应环境线索的作用尚不清楚。我们的初步数据表明，昼夜节律振荡器在小鼠和人类肺部的存在，并进一步表明，这种时钟振荡被香烟烟雾（CS）和COPD患者。我们的初步数据进一步表明，BMAL 1和Per 2的乙酰化在暴露于CS的小鼠的支气管上皮细胞、巨噬细胞和肺中以及在来自COPD患者的痰和肺细胞中增加，与脱乙酰酶sirtuin 1（SIRT 1）的活性/水平的降低相关。SIRT 1的基因消融导致与夸大的肺部炎症反应和肺功能下降相关的昼夜节律钟的抑制，表明SIRT 1在肺昼夜生理学中起着调节作用。然而，CS改变肺部昼夜节律的功能后果尚不清楚。因此，我们假设CS通过下调SIRT 1改变了昼夜节律蛋白BMAL 1和Per 2，导致COPD/肺气肿及其加重小鼠模型中与肺功能下降相关的昼夜节律破坏、肺部炎症增加和类固醇抵抗。我们提出了三个具体的目标，以测试这一假设在体外支气管上皮细胞和巨噬细胞和在体内暴露于CS的小鼠肺。我们建议：（1）确定CS暴露导致人支气管上皮细胞、巨噬细胞和小鼠肺中昼夜节律蛋白BMAL 1和Per 2改变的分子机制;（2）确定SIRT 1通过CS调节BMAL 1、炎症反应和昼夜节律周期的机制;和（3）确定在COPD及其恶化的小鼠模型中，昼夜节律蛋白和SIRT 1对CS介导的昼夜节律周期性、炎症和类固醇抗性的改变的作用，以及COPD急性加重患者。总的来说，这项研究将了解外周昼夜节律耦合肺功能的细胞和分子机制，并确定SIRT 1作为昼夜节律蛋白的战略性时序操纵和类固醇耐药性逆转的新靶点。 (End摘要）

DESCRIPTION (provided by applicant): Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of chronic morbidity and mortality in the United States. As COPD progresses, patients develop more frequent and severe exacerbation, and have an increased rate of emergency room visits and hospitalizations, particularly in the night time and early hours of the day when lung function is lowest and steroids and bronchodilators have their smallest effects. Patients with COPD have abnormal circadian rhythm, reflected in circadian changes in the airway caliber and resistance and respiratory symptoms. However, the cellular and molecular mechanism that underlies altered circadian rhythm in lungs of patients with COPD is not understood. Circadian clock period proteins (Per) and cryptochrome (Cry) and their transcriptional activators core CLOCK and BMAL1 regulate intrinsic daily rhythm, but the role of these molecular oscillators in lung physiology and pathology in response to environmental cues is not known. Our preliminary data show the presence of circadian oscillator in mouse and human lungs and further show that this clock oscillation is disturbed by cigarette smoke (CS) and in patients with COPD. Our preliminary data further show that the acetylation of BMAL1 and Per2 is increased in bronchial epithelial cells, macrophages and lungs of mice exposed to CS, and in sputum and lung cells from COPD patients associated with reduction in the activity/level of deacetylase sirtuin 1 (SIRT1). Genetic ablation of SIRT1 leads to dampening of circadian clock associated with exaggerated lung inflammatory responses and decline in lung function, suggesting that SIRT1 plays a regulatory role in lung circadian physiology. However, the functional consequence of altered circadian rhythmicity in lungs by CS is not known. We, therefore, hypothesize that circadian proteins BMAL1 and Per2 are altered by CS via downregulation of SIRT1 leading to disruption of circadian rhythm, increased lung inflammation and steroid resistance associated with decline in lung function in mouse model of COPD/emphysema and its exacerbations. We propose the three specific aims to test this hypothesis in vitro in bronchial epithelial cells and macrophages and in vivo in mouse lungs exposed to CS. We propose to: (1) determine the molecular mechanisms by which CS exposure results in alteration of circadian proteins BMAL1 and Per 2 in human bronchial epithelial cells, macrophages and in mouse lungs; (2) determine the mechanisms whereby SIRT1 regulates BMAL1, inflammatory response and circadian periodicity by CS; and (3) determine the role of circadian proteins and SIRT1 on CS-mediated alteration in circadian periodicity, inflammation and steroid resistance in a mouse model of COPD and its exacerbations, and in patients with exacerbations of COPD. Overall, this study will understand the cellular and molecular mechanisms of peripheral circadian-coupled lung functions and identify SIRT1 as novel target for strategic chronotherapeutic manipulation of circadian proteins and reversal of steroid resistance. (End of Abstract)