Mettl3 Mediated m6A Methylation Involved in Epithelial-Mesenchymal Transition by Targeting SOCS3/STAT3/SNAI1 in Cigarette Smoking-Induced COPD.

Purpose: Persistent inflammation and epithelial-mesenchymal transition are essential pathophysiological processes in chronic obstructive pulmonary disease (COPD) and involve airway remodeling. m6A methylation modification was discovered to play an important role in various diseases. Nevertheless, the regulatory role of m6A methylation has not yet been investigated in cigarette smoking-induced COPD. The study aims to explore the regulatory role of m6A methylation in cigarette smoking-induced COPD. Patients and Methods: In this study, two Gene Expression Omnibus (GEO) datasets were first utilized to analyze the expression profiles of m6A RNA methylation regulators in COPD. We then established a cell model of COPD by exposing human bronchial epithelial cells (HBECs) to cigarette smoke extract (CSE) in vitro and detected the expression of m6A writer Mettl3 and EMT phenotype markers. RNA interference, cycloleucine, RT-qPCR, western blot, MeRIP-sequencing, and cell migration assay were performed to investigate the potential effect of Mettl3 on the EMT process in CSE-induced HBECs. Results: Our results showed that Mettl3 expression was significantly elevated in cigarette smoking-induced COPD patients and in a cellular model of COPD. Furthermore, Mettl3 silence and cycloleucine treatment inhibited the EMT process of HBECs caused by CSE. Mechanically, Mettl3 silence weakens the m6A methylation of SOCS3 mRNA to enhance the protein expression of SOCS3, inhibiting CSE-induced SOCS3/STAT3/SNAI1 signaling and EMT processes in HBECs. Conclusion: Our study inferred that Mettl3-mediated m6A RNA methylation modification modulates CSE-induced EMT by targeting SOCS3 mRNA and ultimately serves as a crucial regulator in the emergence of COPD. This conclusion reinforces the regulatory role of m6A methylation in COPD.

Exosomes derived from adipose-derived stem cells alleviate cigarette smoke-induced lung inflammation and injury by inhibiting alveolar macrophages pyroptosis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a frequently encountered disease condition in clinical practice mainly caused by cigarette smoke (CS). The aim of this study was to investigate the protective roles of human adipose-derived stem cells-derived exosomes (ADSCs-Exo) in CS-induced lung inflammation and injury and explore the underlying mechanism by discovering the effects of ADSCs-Exo on alveolar macrophages (AMs) pyroptosis. METHODS: ADSCs were isolated from human adipose tissues harvested from three healthy donors, and then ADSCs-Exo were isolated. In vivo, 24 age-matched male C57BL/6 mice were exposed to CS for 4 weeks, followed by intratracheal administration of ADSCs-Exo or phosphate buffered saline. In vitro, MH-S cells, derived from mouse AMs, were stimulated by 2% CS extract (CSE) for 24 h, followed by the treatment of ADSCs-Exo or phosphate buffered saline. Pulmonary inflammation was analyzed by detecting pro-inflammatory cells and mediators in the bronchoalveolar lavage fluid. Lung histology was assessed by hematoxylin and eosin staining. Mucus production was determined by Alcian blue-periodic acid-Schiff staining. The profile of AMs pyroptosis was evaluated by detecting the levels of pyroptosis-indicated proteins. The inflammatory response in AMs and the phagocytic activity of AMs were also investigated. RESULTS: In mice exposed to CS, the levels of pro-inflammatory cells and mediators were significantly increased, mucus production was markedly increased and lung architecture was obviously disrupted. AMs pyroptosis was elevated and AMs phagocytosis was inhibited. However, the administration of ADSCs-Exo greatly reversed these alterations caused by CS exposure. Consistently, in MH-S cells with CSE-induced properties modelling those found in COPD, the cellular inflammatory response was elevated, the pyroptotic activity was upregulated while the phagocytosis was decreased. Nonetheless, these abnormalities were remarkably alleviated by the treatment of ADSCs-Exo. CONCLUSIONS: ADSCs-Exo effectively attenuate CS-induced airway mucus overproduction, lung inflammation and injury by inhibiting AMs pyroptosis. Therefore, hADSCs-Exo may be a promising cell-free therapeutic candidate for CS-induced lung inflammation and injury.

Smoking induces sex-specific changes in the small airway proteome.

INTRODUCTION: Cigarette smoke triggers many cellular and signaling responses in the lung and the resulting inflammation plays a central role in smoke-related lung diseases, such as COPD. We explored the effects of smoking on the small airway proteome in samples obtained by collection of exhaled particles with the aim to identify specific proteins dysregulated by smoking. METHODS: Exhaled particles were obtained from 38 current smokers, 47 former smokers and 22 healthy controls with the PExA method. 120 ng of sample was collected from individual subjects and analyzed with the SOMAscan proteomics platform. General linear model-based statistics were performed. RESULTS: Two hundred and three proteins were detected in at least half of 107 total samples. Active smoking exerted a significant impact on the protein composition of respiratory tract lining fluid (RTLF), with 81 proteins altered in current smokers compared to never smokers (p < 0.05, q < 0.124). Among the proteins most clearly discriminating between current and never smokers were sRAGE, FSTL3, SPOCK2 and protein S, all of them being less abundant in current smokers. Analysis stratified for sex unveiled sex differences with more pronounced proteomic alterations due to active smoking in females than males. Proteins whose abundance was altered by active smoking in women were to a larger extent related to the complement system. The small airway protein profile of former smokers appeared to be more similar to that observed in never smokers. CONCLUSIONS: The study shows that smoking has a strong impact on protein expression in the small airways, and that smoking affects men and women differently, suggesting PExA sampling combined with high sensitivity protein analysis offers a promising platform for early detection of COPD and identification of novel COPD drug targets.

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.

More than just statics: Temporal dynamic changes of intrinsic brain activity in cigarette smoking.

Smoking is companied with altered intrinsic activity of the brain measured by amplitude of low-frequency fluctuation. Evidence has revealed that human brain activity is a highly dynamic and rapidly changing system. How exactly cigarette smoking affect temporal dynamic intrinsic brain activity is not fully understood nor is it clear how smoking severity influences spontaneous brain activity. Dynamic amplitude of low-frequency fluctuation (dALFF) was used to examine the dynamic temporal variability in 93 participants (63 smokers, 30 nonsmokers). We further divided smokers into light and heavy smokers. The temporal variability in intrinsic brain activity among these groups was compared. Correlation analyses were performed between dALFF in areas showing group differences and smoking behaviour (e.g., the Fagerström Test for Nicotine Dependence [FTND] scores and pack-years). Smokers showed significantly increased dALFF in the left inferior/middle frontal gyrus, right orbitofrontal gyrus, right insula, left superior/medial frontal gyrus and right middle frontal gyrus than nonsmokers. Light smokers showed increased dALFF variability in the left prefrontal cortex. Heavy smokers showed increased dynamics in specific brain regions, including the right postcentral gyrus, right insula and left precentral gyrus. Furthermore, the temporal variability in dALFF in the left superior/medial frontal gyrus, left superior/middle frontal gyrus, right middle frontal gyrus and right insula was positively correlated with pack-years or FTND. Combined, these results suggest that smokers increase stable and persistent spontaneous brain activity in prefrontal cortex, involved impaired gold-directed action and value-based decision-making. In addition, individuals with heavier smoking severity show increased perturbance on spontaneous brain activity of perception and sensorimotor, related to increased reliance.

Structural and functional remodeling of the female Apoe-/- mouse aorta due to chronic cigarette smoke exposure.

Despite a decline in popularity over the past several decades, cigarette smoking remains a leading cause of cardiovascular morbidity and mortality. Yet, the effects of cigarette smoking on vascular structure and function are largely unknown. To evaluate changes in the mechanical properties of the aorta that occur with chronic smoking, we exposed female apolipoprotein E-deficient mice to mainstream cigarette smoke daily for 24 wk, with room air as control. By the time of euthanasia, cigarette-exposed mice had lower body mass but experienced larger systolic/diastolic blood pressure when compared with controls. Smoking was associated with significant wall thickening, reduced axial stretch, and circumferential material softening of the aorta. Although this contributed to maintaining intrinsic tissue stiffness at control levels despite larger pressure loads, the structural stiffness became significantly larger. Furthermore, the aorta from cigarette-exposed mice exhibited decreased ability to store elastic energy and augment diastolic blood flow. Histological analysis revealed a region-dependent increase in the cross-sectional area due to smoking. Increased smooth muscle and extracellular matrix content led to medial thickening in the ascending aorta, whereas collagen deposition increased the thickness of the descending thoracic and abdominal aorta. Atherosclerotic lesions were larger in exposed vessels and featured a necrotic core overlaid by a thinned fibrous cap and macrophage infiltration, consistent with a vulnerable phenotype. Collectively, our data indicate that cigarette smoking decreases the mechanical functionality of the aorta, inflicts morphometric alterations to distinct segments of the aorta, and accelerates the progression of atherosclerosis.NEW & NOTEWORTHY We studied the effects of chronic cigarette smoking on the structure and function of the aorta in a mouse model of nose-only aerosol inhalation. Our data indicated that exposure to cigarette smoke impairs vascular function by reducing the ability of the aorta to store elastic energy and by decreasing aortic distensibility. Combined with a more vulnerable atherosclerotic phenotype, these findings reveal the biomechanical mechanisms that support the development of cardiovascular disease due to cigarette smoking.

Eurotium cristatum Fermented Loose Dark Tea Ameliorates Cigarette Smoke-Induced Lung Injury by MAPK Pathway and Enhances Hepatic Metabolic Detoxification by PXR/AhR Pathway in Mice.

Cigarette smoke- (CS-) induced oxidative stress and inflammation in the lung are serious health problems. Primary and reprocessed tea products contain multiple antioxidants that have been reported to protect the lung against CS-induced injury. However, the beneficial effects of Eurotium cristatum fermented loose dark tea (ECT) and Eurotium cristatum particle metabolites (ECP) on CS-induced lung injury and its potential hepatic metabolic detoxification are still unclear. Therefore, sixty mice were randomly divided into six equal groups. CS-exposed mice were prevented or treated with ECP or ECT infusions for 12 or 8 weeks to determine the antioxidative stress, anti-inflammatory and potential metabolic detoxification of ECT and ECP. Thirty-six mice were randomly divided into six equal groups to observe the effects on hepatic metabolic detoxification by replacing daily drinking water with ECT. Results showed that CS significantly decreased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and upregulated the expressions of malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-1ß in serum. These adverse effects were modulated by ECP and ECT. In addition, ECT upregulated the mRNA expression of pregnane X receptor (PXR) and cytochrome P450 (CYP450) in the liver on daily free drinking ECT mice group. Western blot analysis further revealed that in CS-exposed mice, ECP and ECT significantly decreased the phosphorylation of mitogen-activated protein kinase (MAPK) in the lung but upregulated the protein expressions of PXR and aryl hydrocarbon receptor (AhR) in the liver. Overall, our findings demonstrated that ECT and ECP protected against lung injury induced by CS via MAPK pathway and enhanced hepatic metabolic detoxification via PXR and AhR pathways. Therefore, daily intake of ECT and ECP can potentially protect against CS-induced oxidative and inflammatory injuries.

Application of text mining to develop AOP-based mucus hypersecretion genesets and confirmation with in vitro and clinical samples.

Mucus hypersecretion contributes to lung function impairment observed in COPD (chronic obstructive pulmonary disease), a tobacco smoking-related disease. A detailed mucus hypersecretion adverse outcome pathway (AOP) has been constructed from literature reviews, experimental and clinical data, mapping key events (KEs) across biological organisational hierarchy leading to an adverse outcome. AOPs can guide the development of biomarkers that are potentially predictive of diseases and support the assessment frameworks of nicotine products including electronic cigarettes. Here, we describe a method employing manual literature curation supported by a focused automated text mining approach to identify genes involved in 5 KEs contributing to decreased lung function observed in tobacco-related COPD. KE genesets were subsequently confirmed by unsupervised clustering against 3 different transcriptomic datasets including (1) in vitro acute cigarette smoke and e-cigarette aerosol exposure, (2) in vitro repeated incubation with IL-13, and (3) lung biopsies from COPD and healthy patients. The 5 KE genesets were demonstrated to be predictive of cigarette smoke exposure and mucus hypersecretion in vitro, and less conclusively predict the COPD status of lung biopsies. In conclusion, using a focused automated text mining and curation approach with experimental and clinical data supports the development of risk assessment strategies utilising AOPs.

The effect of long-term doxycycline treatment in a mouse model of cigarette smoke-induced emphysema and pulmonary hypertension.

Chronic obstructive pulmonary disease (COPD) is a major cause of death and a still incurable disease, comprising emphysema and chronic bronchitis. In addition to airflow limitation, patients with COPD can suffer from pulmonary hypertension (PH). Doxycycline, an antibiotic from the tetracycline family, in addition to its pronounced antimicrobial activity, acts as a matrix metalloproteinase (MMP) inhibitor and has anti-inflammatory properties. Furthermore, doxycycline treatment exhibited a beneficial effect in several preclinical cardiovascular disease models. In preclinical research, doxycycline is frequently employed for gene expression modulation in Tet-On/Tet-Off transgenic animal models. Therefore, it is crucial to know whether doxycycline treatment in Tet-On/Tet-Off systems has effects independent of gene expression modulation by such systems. Against this background, we assessed the possible curative effects of long-term doxycycline administration in a mouse model of chronic CS exposure. Animals were exposed to cigarette smoke (CS) for 8 mo and then subsequently treated with doxycycline for additional 3 mo in room air conditions. Doxycycline decreased the expression of MMPs and general pro-inflammatory markers in the lungs from CS-exposed mice. This downregulation was, however, insufficient to ameliorate CS-induced emphysema or PH. Tet-On/Tet-Off induction by doxycycline in such models is a feasible genetic approach to study curative effects at least in established CS-induced emphysema and PH. However, we report several parameters that are influenced by doxycycline and use of a Tet-On/Tet-Off system when evaluating those parameters should be interpreted with caution.

Inhalation exposure by cigarette smoke: Effects on the progression of bleomycin- and lipopolysaccharide-induced lung injuries in rat models.

The toxic substances of cigarette smoke (CS) induce inflammatory responses in the lung by recruiting inflammatory cells. In this study, we investigated the effects of CS on the progression of lung disease in bleomycin (BLM) and lipopolysaccharide (LPS)-induced lung injury rat models. Briefly, rats were exposed to CS via inhalation (nose-only) for 28 consecutive days, for 4 h per day. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1) or 0.5 mg/kg LPS (day 26), prepared in 50 µL phosphate-buffered saline (PBS) solution. Examination of the bronchoalveolar lavage fluid (BALF) revealed that the number of neutrophils increased in a concentration-dependent manner of CS. Exposure to CS also enhanced the expression of cytokines, i.e., CCL2 (MCP-1), CCL3 (MIP-1α), CXCL2 (CINC3), CXCL10 (IP-10), TNF-α, IFN-γ, IL-2, IL-4 in the BALF of the vehicle (VC) and BLM groups in a concentration-dependent manner. In particular, the expressions of CCL2, CXCL10 and TNF-α were remarkably upregulated in the BLM + CS 300 treatment as compared to VC, while there were no differences in these cytokine levels in the serum following CS exposure. Exposure to CS resulted in compacted alveolar spaces and macrophage aggregation in the lung tissues following BLM and LPS treatments. Compared to VC, pulmonary fibrosis and chronic inflammation of bronchioloalveoli were observed in the BLM + CS treatment and inflammatory cell infiltration of bronchioloalveoli was observed in the LPS + CS treatment in a concentration-dependent manner by CS. The expression levels of CCL2 and IFN-γ in the lung tissues were increased similar to the levels obtained in BALF, in a concentration-dependent manner by CS. Taken together, these results indicate that repeated exposure to CS may exacerbate the lung injury initially caused by BLM and LPS.

Cigarette smoke preparations, not electronic nicotine delivery system preparations, induce features of lung disease in a 3D lung repeat-dose model.

Cigarette smoking is a risk factor for several lung diseases, including chronic obstructive pulmonary disease, cardiovascular disease, and lung cancer. The potential health effects of chronic use of electronic nicotine delivery systems (ENDS) is unclear. This study utilized fully differentiated primary normal human bronchial epithelial (NHBE) cultures in a repeat-dose exposure to evaluate and compare the effect of combustible cigarette and ENDS preparations. We show that 1-h daily exposure of NHBE cultures over a 10-day period to combustible cigarette whole smoke-conditioned media (WS-CM) increased expression of oxidative stress markers, cell proliferation, airway remodeling, and cellular transformation markers and decreased mucociliary function including ion channel function and airway surface liquid. Conversely, aerosol conditioned media (ACM) from ENDS with similar nicotine concentration (equivalent-nicotine units) as WS-CM and nicotine alone had no effect on those parameters. In conclusion, primary NHBE cultures in a repeat-dose exposure system represent a good model to assess the features of lung disease. This study also reveals that cigarette and ENDS preparations differentially elicit several key endpoints, some of which are potential biomarkers for lung cancer or chronic obstructive pulmonary disease (COPD).

Substantia Nigra Abnormalities Provide New Insight on the Neural Mechanisms Underlying the Sleep-Arousal Phase Dysfunctions in Sudden Infant Death Syndrome.

The purpose of this study was to research possible developmental alterations of the substantia nigra (SN) in sudden infant death syndrome (SIDS), a syndrome frequently attributed to arousal failure from sleep. Brain stems of 46 victims of sudden infant death, aged from 1 to about 7 months (4 to 30 postnatal weeks), were investigated. Twenty-six of these cases were diagnosed as SIDS, due to the lack of any pathological finding, while the remaining 20 cases in which the cause of death was determined at autopsy served as controls. Maternal smoking was reported in 77% of SIDS and 10% of controls. Histopathological examination of the SN was done on 5-µm-thick sections of caudal midbrain stained with both hematoxylin-eosin and Klüver-Barrera. Densitometry, immunohistochemistry and histochemistry were applied to highlight the neuronal concentration, the tyrosine hydroxylase (TH) expression, and the presence of neuromelanin (NM) in this structure. Hypoplasia of the pars compacta portion of the SN was observed in 69% of SIDS but never in controls; TH expression was significantly higher in controls than in SIDS; and NM was observed only in 4 infants of the control group but not in SIDS. A significant correlation was found between SIDS, hypoplasia/low neuronal density, low TH expression in the pars compacta, and maternal smoking. Because the SN pars compacta, being the major dopamine brain center, controls many functions, including the sleep-arousal phase, its alterations, especially concurrently with smoking exposure, may contribute to explain the pathogenesis of SIDS that occur in the great part of cases at awakening from sleep.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,ß-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke.

Exposure to secondhand cigarette smoke is associated with the development of diverse diseases. Resistance training has been considered one of the most useful tools for patients with pulmonary disease, improving their quality of life. This study aimed to evaluate the effect of resistance training (RT) on the prevention of thickening of the right ventricle wall of rats exposed to secondhand cigarette smoke. Thirty-two Wistar rats were divided into four groups: Control (C), Smoker (S), Exercised (E) and Exercised Smoker (ES). The smoker groups were exposed to the smoke of four cigarettes for 30 min, twice daily, five days a week, for 16 weeks. The exercised groups climbed on a vertical ladder with progressive load, once a day, five days a week, for 16 weeks. The heart, trachea, lung, liver and gastrocnemius muscle were removed for histopathological analysis. Pulmonary emphysema (S and ES vs C and E, P < 0.0001) and pulmonary artery thickness enlargement (S vs C and E, P = 0.003, ES vs C, P = 0.003) were detected in the smoking groups. There was an increase in the right ventricle thickness in the S group compared with all other groups (P < 0.0001). An increase in resident macrophages in the liver was detected in both smoking groups compared with the C group (P = 0.002). Additionally, a relevant reduction of the diameter of the muscle fibers was detected only in ES compared with the C, S and E groups (P = 0.0002), impairing, at least in part, the muscle mass in exercised smoking rats. Therefore, it was concluded that resistance training prevented the increase of thickness of the right ventricle in rats exposed to secondhand cigarette smoke, but it may be not so beneficial for the skeletal muscle of smoking rats.

The modulating impact of cigarette smoking on brain structure in panic disorder: a voxel-based morphometry study.

Cigarette smoking increases the likelihood of developing anxiety disorders, among them panic disorder (PD). While brain structures altered by smoking partly overlap with morphological changes identified in PD, the modulating impact of smoking as a potential confounder on structural alterations in PD has not yet been addressed. In total, 143 PD patients (71 smokers) and 178 healthy controls (62 smokers) participated in a multicenter magnetic resonance imaging (MRI) study. T1-weighted images were used to examine brain structural alterations using voxel-based morphometry in a priori defined regions of the defensive system network. PD was associated with gray matter volume reductions in the amygdala and hippocampus. This difference was driven by non-smokers and absent in smoking subjects. Bilateral amygdala volumes were reduced with increasing health burden (neither PD nor smoking > either PD or smoking > both PD and smoking). As smoking can narrow or diminish commonly observed structural abnormalities in PD, the effect of smoking should be considered in MRI studies focusing on patients with pathological forms of fear and anxiety. Future studies are needed to determine if smoking may increase the risk for subsequent psychopathology via brain functional or structural alterations.

Autophagy in chronic lung disease.

The development of chronic lung disease occurs as a consequence of multiple cellular events that involve an initial insult which often leads to the development of chronic inflammation, and the dysregulation of cellular proliferation and cell death mechanisms. Multiple cell types in the lung are key to the respiratory and protective/barrier functions necessary to manage the chronic exposures to environmental, mechanical, and oxidative stressors. Autophagy is essential to lung development and homeostasis, as well as the prevention and development of disease. The cellular process involves the collection and removal of unwanted organelles and proteins through lysosomal degradation. In recent years, investigations have addressed the roles of autophagy and selective autophagy in numerous chronic lung diseases. Here, we highlight recent advances on the role of autophagy in the pathogenesis of asthma, chronic obstructive pulmonary disease and emphysema, pulmonary arterial hypertension, and idiopathic pulmonary fibrosis.

Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers.

BACKGROUND: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs). STUDY DESIGN AND METHODS: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards. RESULTS: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms. CONCLUSION: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy.

CD147 Promoted Epithelial Mesenchymal Transition in Airway Epithelial Cells Induced by Cigarette Smoke via Oxidative Stress Signaling Pathway.

Chronic obstructive pulmonary disease (COPD) is a common airway disease, and epithelial mesenchymal transition (EMT) is participated in the pathogenesis of COPD. However, the role of CD147 in COPD remains largely unknown. In order to clarify the role of CD147 in EMT induced by cigarette smoke, we established animal and cell model of EMT by mean of cigarette smoke exposure and detected the expressions of CD147 and EMT markers via PCR, WB and IF. RNA inference was applied to study the role of CD147 in CSE induced EMT in vitro. NAC and H2O2 were used to study oxidative stress signaling pathway in this model. As a result, cigarette smoke exposure upregulated the expressions of CD147, α-SMA, and Vimentin and downregulated the expression of Ecadherin and ZO1 both in vivo and in vitro, which was accompanied by augmented level of oxidative stress. CD147 knockdown would partly inhibit CSE induced EMT, while preincubation of H2O2 could inverse this effect. In conclusion, CD147 promoted EMT in mice and HBE cells induced by cigarette smoke via oxidative stress signaling pathway.

Evaluation of the acute effects of cigarette smoking on the eye of non-Smoking healthy young male subjects by optical coherence tomography angiography.

Purpose: Investigation of the acute effects of cigarette smoking on the eye using optical coherence tomography angiography (OCTA) on young healthy non-smoking male subjects.Materials and methods: The right eye of 25 healthy non-smoking male subjects were included in this study. Selected parameters of the macular and circumpapillary vasculature were measured, including macular flow indices, foveal avascular zone, vessel densities of macula and optic disc using OCTA. The measurements of the subjects were performed on the first day without smoking at 0 min, 5 min, 30 min and 60 min. Measurements were repeated at the same regime on the second day after smoking. Paired samples t-test and repeated measures one-way analysis of variance test was used for the statistical analysis.Results: The repeated measures analysis of variance test using the Bonferroni adjustment demonstrated a significant change for superficial and deep foveal vessel density, flow area of choriocapillaris, and circumpapillary capillary density following smoking (range of p value was <0.001-0.004). Intraocular pressure, foveal avascular zone area, flow area of outer retina and foveal non-flow area of superficial layer measurements did not have any significant changes following smoking (range of p value was 0.192-0.812).Conclusion: In non-smoking cases, even smoking only one cigarette significantly decreased the vessel density parameters at the acute period. Even though there was an increase in measured parameters shortly after smoking, it had lessened when compared to non-smokers, even one hour following smoking.

Localization of Macrophages in the Human Lung via Design-based Stereology.

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.