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)是美国慢性发病率和死亡率的第四大原因。随着慢性阻塞性肺疾病的进展，患者的病情恶化更加频繁和严重，急诊室就诊和住院率增加，特别是在夜间和凌晨，此时肺功能最差，类固醇和支气管扩张剂的影响最小。COPD患者存在昼夜节律异常，反映在呼吸道管径和阻力的昼夜节律变化和呼吸道症状上。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。昼夜节律时钟周期蛋白(PER)和隐花色素(Cry)及其转录激活子核心时钟和BMAL1调节内在的每日节律，但这些分子振荡器在肺生理和病理中对环境提示的响应作用尚不清楚。我们的初步数据显示，在小鼠和人的肺中存在昼夜节律振荡器，并进一步表明这种时钟振荡受到香烟烟雾(CS)和COPD患者的干扰。我们的初步数据进一步表明，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COPD患者的痰和肺细胞中，BMAL1和PER2的乙酰化水平增加，与脱乙酰酶sirtuin 1(SIRT1)的活性/水平降低有关。基因消融SIRT1可导致与肺炎性反应过度相关的生物钟抑制和肺功能下降，提示SIRT1在肺生理昼夜节律中起调节作用。然而，CS引起的肺部昼夜节律改变的功能后果尚不清楚。因此，我们假设，在COPD/肺气肿及其加重的小鼠模型中，CS通过下调SIRT1而改变了昼夜节律蛋白BMAL1和PER2，导致昼夜节律紊乱、肺部炎症增加和与肺功能下降相关的类固醇抵抗增加。我们提出了三个特定的目标，在体外用支气管上皮细胞和巨噬细胞验证这一假说，并在体内用CS暴露的小鼠肺验证这一假说。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BMAL1和PER_2昼夜节律蛋白改变的分子机制；(2)确定SIRT1调节CS引起的BMAL1、炎症反应和昼夜节律周期性变化的机制；以及(3)确定昼夜节律蛋白和SIRT1在CS介导的COPD及其加重小鼠模型和COPD恶化患者的昼夜节律改变、炎症和类固醇抵抗中的作用。总体而言，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，并将SIRT1确定为昼夜节律蛋白的战略性时间治疗操作和类固醇耐药性逆转的新靶点。 (摘要结束)

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)