p16-3MR: A Novel Model to Study Cellular Senescence in Cigarette Smoke-Induced Lung Injuries.

Cellular senescence and lung aging are associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). COPD progresses with aging, and chronic smoking is the key susceptibility factor in lung pathological changes concurrent with mitochondrial dysfunction and biological aging. However, these processes involving cigarette smoke (CS)-mediated lung cellular senescence are difficult to distinguish. One of the impediments to studying cellular senescence in relation to age-related lung pathologies is the lack of a suitable in vivo model. In view of this, we provide evidence that supports the suitability of p16-3MR mice to studying cellular senescence in CS-mediated and age-related lung pathologies. p16-3MR mice have a trimodal reporter fused to the promoter of the p16INK4a gene that enables detection, isolation, and selective elimination of senescent cells, thus making them a suitable model to study cellular senescence. To determine their suitability in CS-mediated lung pathologies, we exposed young (12-14 months) and old (17-20 months) p16-3MR mice to 30 day CS exposure and studied the expression of senescent genes (p16, p21, and p53) and SASP-associated markers (MMP9, MMP12, PAI-1, and FN-1) in air- and CS-exposed mouse lungs. Our results showed that this model could detect cellular senescence using luminescence and isolate cells undergoing senescence with the help of tissue fluorescence in CS-exposed young and old mice. Our results from the expression of senescence markers and SASP-associated genes in CS-exposed young and old p16-3MR mice were comparable with increased lung cellular senescence and SASP in COPD. We further showed alteration in the; (i) tissue luminescence and fluorescence, (ii) mRNA and protein expressions of senescent markers and SASP genes, and (iii) SA-ß-gal activity in CS-exposed young and old p16-3MR mice as compared to their air controls. Overall, we showed that p16-3MR is a competent model for studying the cellular senescence in CS-induced pathologies. Hence, the p16-3MR reporter mouse model may be used as a novel tool for understanding the pathobiology of cellular senescence and other underlying mechanisms involved in COPD and fibrosis.

Molecular clock REV-ERBα regulates cigarette smoke-induced pulmonary inflammation and epithelial-mesenchymal transition.

Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/chronic obstructive pulmonary disease (COPD), which is associated with abnormal epithelial-mesenchymal transition (EMT). Previously, we have shown an association among circadian rhythms, CS-induced lung inflammation, and nuclear heme receptor α (REV-ERBα), acting as an antiinflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J WT and REV-ERBα heterozygous (Het) and -KO mice were exposed to CS for 30 days (subchronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Subchronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα-KO mice; both circadian and EMT dysregulation were exaggerated in the REV-ERBα-KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Moreover, REV-ERBα agonist (GSK4112) inhibited TGF-ß/CS-induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1). Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα-dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.

Prenatal Exposure to Electronic-Cigarette Aerosols Leads to Sex-Dependent Pulmonary Extracellular-Matrix Remodeling and Myogenesis in Offspring Mice.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Age-dependent assessment of genes involved in cellular senescence, telomere and mitochondrial pathways in human lung tissue of smokers, COPD and IPF: Associations with SARS-CoV-2 COVID-19 ACE2-TMPRSS2-Furin-DPP4 axis.

Aging is one of the key contributing factors for chronic obstructive pulmonary diseases (COPD) and other chronic inflammatory lung diseases. Cigarette smoke is a major etiological risk factor that has been shown to alter cellular processes involving mitochondrial function, cellular senescence and telomeric length. Here we determined how aging contribute to the alteration in the gene expression of above mentioned cellular processes that play an important role in the progression of COPD and IPF. We hypothesized that aging may differentially alter the expression of mitochondrial, cellular senescence and telomere genes in smokers and patients with COPD and IPF compared to non-smokers. Total RNA from human lung tissues from non-smokers, smokers, and patients with COPD and IPF were processed and analyzed based on their ages (younger: <55 yrs and older: >55 yrs). NanoString nCounter panel was used to analyze the gene expression profiles using a custom designed codeset containing 112 genes including 6 housekeeping controls (mitochondrial biogenesis and function, cellular senescence, telomere replication and maintenance). mRNA counts were normalized, log2 transformed for differential expression analysis using linear models in the limma package (R/Bioconductor). Data from non-smokers, smokers and patients with COPD and IPF were analyzed based on the age groups (pairwise comparisons between younger vs. older groups). Several genes were differentially expressed in younger and older smokers, and patients with COPD and IPF compared to non-smokers which were part of the mitochondrial biogenesis/function (HSPD1, FEN1, COX18, COX10, UCP2 & 3), cellular senescence (PCNA, PTEN, KLOTHO, CDKN1C, TNKS2, NFATC1 & 2, GADD45A) and telomere replication/maintenance (PARP1, SIRT6, NBN, TERT, RAD17, SLX4, HAT1) target genes. Interestingly, NOX4 and TNKS2 were increased in the young IPF as compared to the young COPD patients. Genes in the mitochondrial dynamics and other quality control mechanisms like FIS1 and RHOT2 were decreased in young IPF compared to their age matched COPD subjects. ERCC1 (Excision Repair Cross-Complementation Group 1) and GADD45B were higher in young COPD as compared to IPF. Aging plays an important role in various infectious diseases. Elderly patients with chronic lung disease and smokers were found to have high incidence and mortality rates in the current pandemic of SARS-CoV-2 infection. Immunoblot analysis in the lung homogenates of smokers, COPD and IPF subjects revealed increased protein abundance of important proteases and spike proteins like TMPRSS2, furin and DPP4 in association with a slight increase in SARS-CoV-2 receptor ACE2 levels. This may further strengthen the observation that smokers, COPD and IPF subjects are more prone to COVID-19 infection. Overall, these findings suggest that altered transcription of target genes that regulate mitochondrial function, cellular senescence, and telomere attrition add to the pathobiology of lung aging in COPD and IPF and other smoking-related chronic lung disease in associated with alterations in SARS-CoV-2 ACE2-TMPRSS2-Furin-DPP4 axis for COVID-19 infection.

E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Cellular stress responses and dysfunctional Mitochondrial-cellular senescence, and therapeutics in chronic respiratory diseases.

The abnormal inflammatory responses due to the lung tissue damage and ineffective repair/resolution in response to the inhaled toxicants result in the pathological changes associated with chronic respiratory diseases. Investigation of such pathophysiological mechanisms provides the opportunity to develop the molecular phenotype-specific diagnostic assays and could help in designing the personalized medicine-based therapeutic approaches against these prevalent diseases. As the central hubs of cell metabolism and energetics, mitochondria integrate cellular responses and interorganellar signaling pathways to maintain cellular and extracellular redox status and the cellular senescence that dictate the lung tissue responses. Specifically, as observed in chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, the mitochondria-endoplasmic reticulum (ER) crosstalk is disrupted by the inhaled toxicants such as the combustible and emerging electronic nicotine-delivery system (ENDS) tobacco products. Thus, the recent research efforts have focused on understanding how the mitochondria-ER dysfunctions and oxidative stress responses can be targeted to improve inflammatory and cellular dysfunctions associated with these pathologic illnesses that are exacerbated by viral infections. The present review assesses the importance of these redox signaling and cellular senescence pathways that describe the role of mitochondria and ER on the development and function of lung epithelial responses, highlighting the cause and effect associations that reflect the disease pathogenesis and possible intervention strategies.

Association of smoking and electronic cigarette use with wheezing and related respiratory symptoms in adults: cross-sectional results from the Population Assessment of Tobacco and Health (PATH) study, wave 2.

BACKGROUND: Wheezing is a symptom of potential respiratory disease and known to be associated with smoking. Electronic cigarette use ('vaping') has increased exponentially in recent years. This study examined the cross-sectional association of vaping with wheezing and related respiratory symptoms and compare this association with smokers and dual users. METHODS: The Population Assessment of Tobacco and Health study wave 2 data collected from October 2014 to October 2015 with 28 171 adults were used. The cross-sectional association of vaping with self-reported wheezing and related respiratory symptoms relative to smokers and dual users of tobacco and electronic cigarettes were studied using multivariable logistic and cumulative logistic regression models with consideration of complex sampling design. RESULTS: Among the 28 171 adult participants, 641 (1.2%) were current vapers who used e-cigarettes exclusively, 8525 (16.6%) were current exclusive smokers, 1106 (2.0%) were dual users and 17 899 (80.2%) were non-users. Compared with non-users, risks of wheezing and related respiratory symptoms were significantly increased in current vapers (adjusted OR (aOR)=1.67, 95% CI: 1.23 to 2.15). Current vapers had significantly lower risk in wheezing and related respiratory symptoms compared with current smokers (aOR=0.68, 95% CI: 0.53 to 0.87). No significant differences were found between dual users and current smokers in risk of wheezing and related respiratory symptoms (aOR=1.06, 95% CI: 0.91 to 1.24). CONCLUSIONS: Vaping was associated with increased risk of wheezing and related respiratory symptoms. Current vapers had lower risk in wheezing and related respiratory symptoms than current smokers or dual users but higher than non-users. Both dual use and smoking significantly increased the risk of wheezing and related respiratory symptoms.

Dysregulated repair and inflammatory responses by e-cigarette-derived inhaled nicotine and humectant propylene glycol in a sex-dependent manner in mouse lung.

Nicotine inhalation via electronic cigarettes (e-cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine-containing e-cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 h daily for 3 days to e-cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine-containing e-cig aerosols induced inflammatory cell influx (neutrophils and CD8a+ T-lymphocytes), and release of pro-inflammatory cytokines in bronchoalveolar lavage fluid in a sex-dependent manner. Inhalation of e-cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, ß-catenin, E-cadherin, and MMP2) in a sex-dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α-smooth muscle actin and ß-catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine containing e-cig aerosols or PG alone differentially affected the release of pro-inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine containing e-cig aerosols was sufficient to elicit a pro-inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex-dependent manner. Thus, acute exposure to inhaled nicotine via e-cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs.

Mitochondrial dysfunction is associated with Miro1 reduction in lung epithelial cells by cigarette smoke.

Cigarette smoke (CS) is known to cause mitochondrial dysfunction leading to cellular senescence in lung cells. We determined the mechanism of mitochondrial dysfunction by CS in lung epithelial cells. CS extract (CSE) treatment differentially affected mitochondrial function, such as membrane potential, mitochondrial reactive oxygen species (mtROS) and mitochrondrial mass as analyzed by FACS, and were associated with altered oxidative phosphorylation (OXPHOS) protein levels (Complexes I-IV) in primary lung epithelial cells (SAEC and NHBE), and (complexes I and II) in BEAS2B cells. There were dose- and time-dependent changes in mitochondrial respiration (oxygen consumption rate parameters i.e. maximal respiration, ATP production and spare capacity, measured by the Seahorse analyzer) in control vs. CSE treated BEAS2B and NHBE/DHBE cells. Electron microscopy (EM) analysis revealed perinuclear clustering by localization and increased mitochondrial fragmentation by fragement length analysis. Immunoblot analysis revealed CS-mediated increase in Drp1 and decrease in Mfn2 levels that are involved in mitochondrial fission/fusion process. CSE treatment reduced Miro1 and Pink1 abundance that play a crucial role in the intercellular transfer mechanism and mitophagy process. Overall, these findings highlight the role of Miro1 in context of CS-induced mitochondrial dysfunction in lung epithelial cells that may contribute to the pathogenesis of chronic inflammatory lung diseases.

Waterpipe smoke and e-cigarette vapor differentially affect circadian molecular clock gene expression in mouse lungs.

The use of emerging tobacco products, such as waterpipe or hookah and electronic cigarettes (e-cigs), has gained significant popularity and are promoted as safer alternatives to conventional cigarettes. Circadian systems are internal biological oscillations that are considered important regulators of immune functions in mammals. Tobacco induced inflammatory lung diseases frequently exhibit time-of-day/night variation in lung function and symptom severity. We investigated the impact of inhaled e-cig vapor and waterpipe smoke (WPS) on pulmonary circadian molecular clock disruption by determining the changes in expression levels and abundance of core clock component genes (BMAL1, CLOCK) and clock-controlled output genes (Rev-erbα, Per2, Rev-erbß, Cry2, Rorα) in mouse lungs. We showed that the expression levels of these pulmonary core clock genes and clock-controlled output genes were altered significantly following exposure to WPS (Bmal1, Clock, and Rev-erbα). We further showed a significant yet differential effect on expression levels of core clock and clock-controlled genes (Bmal1, Per2) in the lungs of mice exposed to e-cig vapor containing nicotine. Thus, acute exposure to WPS and e-cig vapor containing nicotine contributes to altered expression of circadian molecular clock genes in mouse lungs, which may have repercussions on lung cellular and biological functions.

Lung cellular senescence is independent of aging in a mouse model of COPD/emphysema.

Cigarette smoke (CS) induces lung cellular senescence that plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How aging influences cellular senescence and other molecular hallmarks, and increases the risk of CS-induced damage remains unknown. We hypothesized that aging-associated changes in lungs worsen the COPD/emphysema by CS exposure. Younger and older groups of C57BL/6J mice were exposed to chronic CS for 6 months with respective age-matched air-exposed controls. CS caused a decline in lung function and affected the lung structure of both groups of mice. No alterations were observed in the induction of inflammatory mediators between the air-exposed younger and older controls, but aging increased the severity of CS-induced lung inflammation. Aging per se increased lung cellular senescence and significant changes in damage-associated molecular patterns marker S100A8. Gene transcript analysis using the nanoString nCounter showed a significant upregulation of key pro-senescence targets by CS (Mmp12, Ccl2, Cdkn2a, Tert, Wrn, and Bub1b). Aging independently influenced lung function and structure, as well as increased susceptibility to CS-induced inflammation in emphysema, but had a negligible effect on cellular senescence. Thus, aging solely does not contribute to the induction of cellular senescence by CS in a mouse model of COPD/emphysema.

Chronic cigarette smoke exposure drives spiral ganglion neuron loss in mice.

Tobacco use is associated with an increased risk of hearing loss in older individuals, suggesting cigarette smoke (CS) exposure may target the peripheral auditory organs. However, the effects of CS exposure on general cochlear anatomy have not previously been explored. Here we compare control and chronic CS exposed cochleae from adult mice to assess changes in structure and cell survival. Two-photon imaging techniques, including the imaging of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) from native molecules, were used to probe the whole cochlear organ for changes. We found evidence for fibrillar collagen accumulation in the spiral ganglion and organ of Corti, consistent with fibrosis. Quantitative TPEF indicated that basal CS-exposed spiral ganglion neurons experienced greater oxidative stress than control neurons, which was confirmed by histological staining for lipid peroxidation products. Cell counts confirmed that the CS-exposed spiral ganglion also contained fewer basal neurons. Taken together, these data support the premise that CS exposure induces oxidative stress in cochlear cells. They also indicate that two-photon techniques may screen cochlear tissues for oxidative stress.

Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Histone deacetylase 2 (HDAC2), a critical determinant of chromatin remodeling, is reduced as a consequence of oxidative stress-mediated DNA damage and impaired repair. Cigarette smoke (CS) exposure causes DNA damage and cellular senescence. However, no information is available on the role of HDAC2 in CS-induced DNA damage, stress-induced premature senescence (SIPS), and senescence-associated secretory phenotype (SASP) during the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that CS causes persistent DNA damage and cellular senescence via HDAC2-dependent mechanisms. We used HDAC2 global knockout (KO) and HDAC2 lung epithelial cell-specific KO [Clara cell-specific HDAC2 deletion (HDAC2 CreCC10)] mice to determine whether HDAC2 is a major player in CS-induced oxidative stress, SIPS, and SASP. HDAC2 KO mice exposed to CS show exaggerated DNA damage, inflammatory response, and decline in lung function leading to airspace enlargement. Chronic CS exposure augments lung senescence-associated ß-galactosidase activity in HDAC2 KO, but not in HDAC2 CreCC10 mice. HDAC2 lung epithelial cell-specific KO did not further augment CS-induced inflammatory response and airspace enlargement but instead caused an increase in lymphoid aggregate formation. Our study reveals that HDAC2 is a key player regulating CS-induced DNA damage, inflammatory response, and cellular senescence leading to COPD/emphysema.-Sundar, I. K., Rashid, K., Gerloff, J., Rangel-Moreno, J., Li, D., Rahman, I. Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Genetic Ablation of p16INK4a Does Not Protect against Cellular Senescence in Mouse Models of Chronic Obstructive Pulmonary Disease/Emphysema.

Cigarette smoke (CS) affects DNA damage and cellular senescence signaling pathways in the pathogenesis of chronic obstructive pulmonary disease (COPD). p16INK4a (p16: a cyclin-dependent kinase inhibitor) is a key marker of cellular senescence, which is induced by CS in lung cells. It is thought that removal of p16 attenuates premature aging by removing senesced cells. However, the role of p16 in CS-induced stress-induced premature senescence (SIPS) and senescence-associated secretory phenotype (SASP) during the development of COPD/emphysema is not known. We hypothesize that p16 regulates cellular senescence and DNA damage/repair molecular signaling targets during chronic CS-induced inflammation and airspace enlargement in mouse models of COPD. We used p16 global knockout (KO) and p16 lung epithelial cell-specific KO (p16CreCC10) mice to determine whether p16 removal in lung epithelium augments or protects against cellular senescence (SIPS and SASP) in chronic CS- and elastase-induced development of COPD/emphysema in mice. p16 KO mice exposed to chronic CS and p16 lung epithelial cell-specific KO mice exposed to elastase did not show attenuation of lung inflammation, altered lung function, or airspace enlargement. p16 KO and p16CreCC10 exposed to CS and elastase showed increases in lung senescence-associated ß-galactosidase activity. Thus, removal of p16-positive cells did not protect against airspace enlargement and decline in lung function induced in COPD mouse models. Our findings suggest that p16 is not the only key player associated with CS-induced cellular senescence phenotypes (SIPS and SASP), decline in lung function, and airspace enlargement in COPD/emphysema.

Redox regulation of circadian molecular clock in chronic airway diseases.

At the cellular level, circadian timing is maintained by the molecular clock, a family of interacting clock gene transcription factors, nuclear receptors and kinases called clock genes. Daily rhythms in pulmonary function are dictated by the circadian timing system, including rhythmic susceptibility to the harmful effects of airborne pollutants, exacerbations in patients with chronic airway disease and the immune-inflammatory response to infection. Further, evidence strongly suggests that the circadian molecular clock has a robust reciprocal interaction with redox signaling and plays a considerable role in the response to oxidative/carbonyl stress. Disruption of the circadian timing system, particularly in airway cells, impairs pulmonary rhythms and lung function, enhances oxidative stress due to airway inhaled pollutants like cigarette smoke and airborne particulate matter and leads to enhanced inflammosenescence, inflammasome activation, DNA damage and fibrosis. Herein, we briefly review recent evidence supporting the role of the lung molecular clock and redox signaling in regulating inflammation, oxidative stress, and DNA damage responses in lung diseases and their exacerbations. We further describe the potential for clock genes as novel biomarkers and therapeutic targets for the treatment of chronic lung diseases.

The nuclear receptor and clock gene REV-ERBα regulates cigarette smoke-induced lung inflammation.

REV-ERBα is a nuclear heme receptor, transcriptional repressor and critical component of the molecular clock that drives daily rhythms of metabolism. Evidence reveals that REV-ERBα also plays an important regulatory role in clock-dependent lung physiology and inflammatory responses. We hypothesize that cigarette smoke (CS) exposure influences REV-ERBα abundance in the lungs, facilitating a pro-inflammatory phenotype. To determine the impact of REV-ERBα activation in the CS-induced inflammatory response we treated primary human small airway epithelial cells (SAECs) with CS extract (CSE) or lipopolysaccharide (LPS) in the absence or presence of pre-treatment with the REV-ERBα agonist GSK 4112. We also exposed adult C57BL/6J (WT) and Rev-erbα global KO mice to CS (10 and 30 days) and measured pro-inflammatory cytokine release. Our data reveal that pre-treatment with GSK 4112 reduced CSE/LPS induced pro-inflammatory cytokines release from both SAECs and mouse lung fibroblasts (MLFs). Furthermore, REV-ERBα KO mice show a greater inflammatory response to 10 and 30 days of CS, including increased neutrophil lung influx, pro-inflammatory cytokine (IL-6, MCP-1 and KC) release, and pro-senescence marker (p16) when compared to WT mice. These data demonstrate that REV-ERBα is a critical regulator of CS-induced lung inflammatory responses.

DNA methylation profiling in peripheral lung tissues of smokers and patients with COPD.

BACKGROUND: Epigenetics changes have been shown to be affected by cigarette smoking. Cigarette smoke (CS)-mediated DNA methylation can potentially affect several cellular and pathophysiological processes, acute exacerbations, and comorbidity in the lungs of patients with chronic obstructive pulmonary disease (COPD). We sought to determine whether genome-wide lung DNA methylation profiles of smokers and patients with COPD were significantly different from non-smokers. We isolated DNA from parenchymal lung tissues of patients including eight lifelong non-smokers, eight current smokers, and eight patients with COPD and analyzed the samples using Illumina's Infinium HumanMethylation450 BeadChip. RESULTS: Our data revealed that the differentially methylated genes were related to top canonical pathways (e.g., G beta gamma signaling, mechanisms of cancer, and nNOS signaling in neurons), disease and disorders (organismal injury and abnormalities, cancer, and respiratory disease), and molecular and cellular functions (cell death and survival, cellular assembly and organization, cellular function and maintenance) in patients with COPD. The genome-wide DNA methylation analysis identified suggestive genes, such as NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7 with DNA methylation changes in COPD lung tissues that were further validated by pyrosequencing. Pyrosequencing validation confirmed hyper-methylation in smokers and patients with COPD as compared to non-smokers. However, we did not detect significant differences in DNA methylation for TNFAIP2, ATXN7, and THOC7 genes in smokers and COPD groups despite the changes observed in the genome-wide analysis. CONCLUSIONS: Our study suggests that DNA methylation in suggestive genes, such as NOS1AP, BID, and GABRB1 may be used as epigenetic signatures in smokers and patients with COPD if the same is validated in a larger cohort. Future studies are required to correlate DNA methylation status with transcriptomics of selective genes identified in this study and elucidate their role and involvement in the progression of COPD and its exacerbations.

Myofibroblast differentiation and its functional properties are inhibited by nicotine and e-cigarette via mitochondrial OXPHOS complex III.

Nicotine is the major stimulant in tobacco products including e-cigarettes. Fibroblast to myofibroblast differentiation is a key process during wound healing and is dysregulated in lung diseases. The role of nicotine and e-cigarette derived nicotine on cellular functions including profibrotic response and other functional aspects is not known. We hypothesized that nicotine and e-cigarettes affect myofibroblast differentiation, gel contraction, and wound healing via mitochondria stress through nicotinic receptor-dependent mechanisms. To test the hypothesis, we exposed human lung fibroblasts with various doses of nicotine and e-cigarette condensate and determined myofibroblast differentiation, mitochondrial oxidative phosphorylation (OXPHOS), wound healing, and gel contraction at different time points. We found that both nicotine and e-cigarette inhibit myofibroblast differentiation as shown by smooth muscle actin and collagen type I, alpha 1 abundance. Nicotine and e-cigarette inhibited OXPHOS complex III accompanied by increased MitoROS, and this effect was augmented by complex III inhibitor antimycin A. These mitochondrial associated effects by nicotine resulted in inhibition of myofibroblast differentiation. These effects were associated with inhibition of wound healing and gel contraction suggesting that nicotine is responsible for dysregulated repair during injurious responses. Thus, our data suggest that nicotine causes dysregulated repair by inhibition of myofibroblast differentiation via OXPHOS pathway.

Inflammatory Response and Barrier Dysfunction by Different e-Cigarette Flavoring Chemicals Identified by Gas Chromatography-Mass Spectrometry in e-Liquids and e-Vapors on Human Lung Epithelial Cells and Fibroblasts.

Recent studies suggest that electronic cigarette (e-cig) flavors can be harmful to lung tissue by imposing oxidative stress and inflammatory responses. The potential inflammatory response by lung epithelial cells and fibroblasts exposed to e-cig flavoring chemicals in addition to other risk-anticipated flavor enhancers inhaled by e-cig users is not known. The goal of this study was to evaluate the release of the proinflammatory cytokine (interleukin-8 [IL-8]) and epithelial barrier function in response to different e-cig flavoring chemicals identified in various e-cig e-liquid flavorings and vapors by chemical characterization using gas chromatography-mass spectrometry analysis. Flavorings, such as acetoin (butter), diacetyl, pentanedione, maltol (malt), ortho-vanillin (vanilla), coumarin, and cinnamaldehyde in comparison with tumor necrosis factor alpha (TNFα), were used in this study. Human bronchial epithelial cells (Beas2B), human mucoepidermoid carcinoma epithelial cells (H292), and human lung fibroblasts (HFL-1) were treated with each flavoring chemical for 24 hours. The cells and conditioned media were then collected and analyzed for toxicity (viability %), lung epithelial barrier function, and proinflammatory cytokine IL-8 release. Cell viability was not significantly affected by any of the flavoring chemicals tested at a concentration of 10 µM to 1 mM. Acetoin and diacetyl treatment induced IL-8 release in Beas2B cells. Acetoin- and pentanedione-treated HFL-1 cells produced a differential, but significant response for IL-8 release compared to controls and TNFα. Flavorings, such as ortho-vanillin and maltol, induced IL-8 release in Beas2B cells, but not in H292 cells. Of all the flavoring chemicals tested, acetoin and maltol were more potent inducers of IL-8 release than TNFα in Beas2B and HFL-1 cells. Flavoring chemicals rapidly impaired epithelial barrier function in human bronchial epithelial cells (16-HBE) as measured by electric cell surface impedance sensing. Our findings suggest that some of the e-cig liquids/aerosols containing flavoring chemicals can cause significant loss of epithelial barrier function and proinflammatory response in lung cells.