Chronic Tobacco Smoke Exposure Research Articles

CSP7 Protects Alveolar Epithelial Cells by Targeting p53-Fibrinolytic Pathways During Lung Injuries.

Impaired alveolar epithelial regeneration in patients with idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) is attributed to telomere dysfunction in type II alveolar epithelial cells (A2Cs). Genetic susceptibility, aging, and toxicant exposures, including tobacco smoke (TS), contribute to telomere dysfunction in A2Cs. Here we investigated whether improvement of telomere function plays a role in CSP7-mediated protection of A2Cs against ongoing senescence and apoptosis during bleomycin (BLM)-induced pulmonary fibrosis (PF) as well as alveolar injury caused by chronic TS exposure. We found a significant telomere shortening in A2Cs isolated from IPF and COPD lungs in line with other studies. These cells showed increased p53 in addition to its post-translational modification with induction of activated caspase-3 and β-galactosidase, suggesting a p53-mediated loss of A2C renewal. Further, we found increased expression of SIAH-1, a p53-inducible E3 ubiquitin ligase known to down-regulate telomere repeats binding factor 2 (TRF2). Consistent with the loss of TRF2 and upregulation of TRF1, telomerase reverse transcriptase (TERT) was downregulated in A2Cs. A2Cs from fibrotic lungs of mice either repeatedly instilled with BLM or isolated from chronic TS exposure-induced lung injury model showed reduced telomere length along with induction of p53, PAI-1, SIAH1 and TRF1 as well as loss of TRF2 and TERT, which were reversed in wild-type mice after treatment with CSP7. Interestingly, PAI-1-/- mice, or those lacking microRNA-34a expression in A2Cs, resisted telomere dysfunction, while uPA-/- mice failed to respond to CSP7 treatment, suggesting p53-microRNA-34a feed-forward induction and p53-uPA pathway contributes to telomere dysfunction.

Impairment of Gal-9 and Tim-3 crosstalk between Tregs and Th17 cells drives tobacco smoke-induced airway inflammation.

Overexpression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells has been observed in smokers. However, whether and how galectin-9 (Gal-9)/TIM-3 signal between T-regulatory cells (Tregs) and type 17 helper (Th17) cells contributes to tobacco smoke-induced airway inflammation remains unclear. Here, we aimed to explore the role of the Gal-9/TIM-3 signal between Tregs and Th17 cells during chronic tobacco smoke exposure. Tregs phenotype and the expression of TIM-3 on CD4+ T cells were detected in a mouse model of experimental emphysema. The role of TIM-3 in CD4+ T cells was explored in a HAVCR2-/- mouse model and in mice that received recombinant anti-TIM3. The crosstalk between Gal-9 and Tim-3 was evaluated by coculture Tregs with effector CD4+ T cells. We also invested the expression of Gal-9 in Tregs in patients with COPD. Our study revealed that chronic tobacco smoke exposure significantly reduces the frequency of Tregs in the lungs of mice and remarkably shapes the heterogeneity of Tregs by downregulating the expression of Gal-9. We observed a pro-inflammatory but restrained phenotypic transition of CD4+ T cells after tobacco smoke exposure, which was maintained by TIM-3. The restrained phenotype of CD4+ T cells was perturbed when TIM-3 was deleted or neutralised. Tregs from the lungs of mice with emphysema displayed a blunt ability to inhibit the differentiation and proliferation of Th17 cells. The inhibitory function of Tregs was partially restored by using recombinant Gal-9. The interaction between Gal-9 and TIM-3 inhibits the differentiation of Th17 cells and promotes apoptosis of CD4+ T cells, possibly by interfering with the expression of retinoic acid receptor-related orphan receptor gamma t. The expression of Gal-9 in Tregs was reduced in patients with COPD, which was associated with Th17 response and lung function. These findings present a new paradigm that impairment of Gal-9/Tim-3 crosstalk between Tregs and Th17 cells during chronic tobacco smoke exposure promotes tobacco smoke-induced airway/lung inflammation.

Therapeutic benefits of quercetin in traumatic brain injury model exposed to cigarette smoke

Scientific evidences reported the deleterious effect of cigarette smoking or passive smoking on brain health particularly cognitive functions, blood–brain barrier (BBB) permeability, up-regulation of inflammatory cascades, and depletion of the antioxidant system. These combined effects become more progressive in the events of stroke, traumatic brain injury (TBI), and many other neurodegenerative diseases. In the current study, we investigated the long-term administered therapeutic potential of quercetin in ameliorating the deleterious neurobiological consequences of chronic tobacco smoke exposure in TBI mice. After exposure to 21 days of cigarette smoke and treatment with 50 mg/kg of quercetin, C57BL/6 mice were challenged for the induction of TBI by the weight drop method. Subsequently, a battery of behavioral tests and immunohistochemical analyses revealed the beneficial effect of quercetin on the locomotive and cognitive function of TBI + smoked group mice (p < 0.05 vs control sham). Immunohistochemistry analysis (Nrf2, HO-1, NFkB, caspase 3) demonstrated a marked protection after 21 days of quercetin treatment in the chronic tobacco smoking group possibly by up-regulation of antioxidant pathways, and decreased apoptosis. In conclusion, our findings support the therapeutic effectiveness of quercetin in partly protecting the central neurological functions that become aberrantly impaired in combined habitual cigarette-smoking individuals impacted with TBI.

Use of Conventional Cigarette Smoking and E-Cigarette Vaping for Experimental Stroke Studies in Mice.

Cigarette smoking is a major prodromal factor for the onset of many adverse health effects that may occur in the short run and is the leading cause of preventable disease, disability, and death in the United States. Moreover, it is well established that chronic smoking is associated with vascular endothelial dysfunction in a causative and dose-dependent manner primarily related to the release of reactive oxygen species (ROS), nicotine, and the induction of oxidative stress (OS)-driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid ingredient used in e-cigarettes) can also cause OS, exacerbating cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to cerebrovascular impairment and promote cerebrovascular conditions favoring stroke onset and worsening post-ischemic brain injury. Therefore, using mouse models is crucial to understand how xenobiotics such as those released by conventional and/or e-cigs can impact the onset and severity of stroke as well as post-stroke recovery. To appropriately model human-like smoking/vaping behavior in mice, however, the exposure to these xenobiotics must be standardized and undertaken in a controlled environment. This chapter describes a well-validated protocol to reproduce standardized chronic tobacco smoke or e-cigarette vape exposure in mice in the setting of a mouse transient ischemic stroke model.

Chronic aryl hydrocarbon receptor activity phenocopies smoking-induced skeletal muscle impairment.

BackgroundChronic obstructive pulmonary disease (COPD) patients exhibit skeletal muscle atrophy, denervation, and reduced mitochondrial oxidative capacity. Whilst chronic tobacco smoke exposure is implicated in COPD muscle impairment, the mechanisms involved are ambiguous. The aryl hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that activates detoxifying pathways with numerous exogenous ligands, including tobacco smoke. Whereas transient AHR activation is adaptive, chronic activation can be toxic. On this basis, we tested the hypothesis that chronic smoke‐induced AHR activation causes adverse muscle impact.MethodsWe used clinical patient muscle samples, and in vitro (C2C12 myotubes) and in vivo models (mouse), to perform gene expression, mitochondrial function, muscle and neuromuscular junction morphology, and genetic manipulations (adeno‐associated virus‐mediated gene transfer).ResultsSixteen weeks of tobacco smoke exposure in mice caused muscle atrophy, neuromuscular junction degeneration, and reduced oxidative capacity. Similarly, smoke exposure reprogrammed the muscle transcriptome, with down‐regulation of mitochondrial and neuromuscular junction genes. In mouse and human patient specimens, smoke exposure increased muscle AHR signalling. Mechanistically, experiments in cultured myotubes demonstrated that smoke condensate activated the AHR, caused mitochondrial impairments, and induced an AHR‐dependent myotube atrophy. Finally, to isolate the role of AHR activity, expression of a constitutively active AHR mutant without smoke exposure caused atrophy and mitochondrial impairments in cultured myotubes, and muscle atrophy and neuromuscular junction degeneration in mice.ConclusionsThese results establish that chronic AHR activity, as occurs in smokers, phenocopies the atrophy, mitochondrial impairment, and neuromuscular junction degeneration caused by chronic tobacco smoke exposure.

An Improved Murine Premalignant Squamous Cell Model: Tobacco Smoke Exposure Augments NTCU-Induced Murine Airway Dysplasia.

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4-5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3-6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development.

Distinguishing Smoking-Related Lung Disease Phenotypes Via Imaging and Molecular Features

Chronic tobacco smoke exposure results in a broad range of lung pathologies including emphysema, airway disease and parenchymal fibrosis as well as a multitude of extra-pulmonary comorbidities. Prior work using CT imaging has identified several clinically relevant subgroups of smoking related lung disease, but these investigations have generally lacked organ specific molecular correlates. Can CT imaging be used to identify clinical phenotypes of smoking related lung disease that have specific bronchial epithelial gene expression patterns to better understand disease pathogenesis? Using K-means clustering, we clustered participants from the COPDGene study (n= 5,273) based on CT imaging characteristics and then evaluated their clinical phenotypes. These clusters were replicated in the Detection of Early Lung Cancer Among Military Personnel (DECAMP) cohort (n= 360), and were further characterized using bronchial epithelial gene expression. Three clusters (preserved, interstitial predominant and emphysema predominant) were identified. Compared to the preserved cluster, the interstitial and emphysema clusters had worse lung function, exercise capacity and quality of life. In longitudinal follow-up, individuals from the emphysema group had greater declines in exercise capacity and lung function, more emphysema, more exacerbations, and higher mortality. Similarly, genes involved in inflammatory pathways (tumor necrosis factor-α, interferon-β) are more highly expressed in bronchial epithelial cells from individuals in the emphysema cluster, while genes associated with T-cell related biology are decreased in these samples. Samples from individuals in the interstitial cluster generally had intermediate levels of expression of these genes. Using quantitative CT imaging, we identified three groups of individuals in older ever-smokers that replicate in two cohorts. Airway gene expression differences between the three groups suggests increased levels of inflammation in the most severe clinical phenotype, possibly mediated by the tumor necrosis factor-α and interferon-β pathways. COPDGene (NCT00608764), DECAMP-1 (NCT01785342), DECAMP-2 (NCT02504697).

Chronic Maternal Tobacco Smoke Exposure and/or Alpha-Lipoic Acid Treatment Causes Long-Term Deterioration of Testis and Sexual Behavior in Adult Male Rats

BackgroundTobacco use during pregnancy is known to have several negative effects on the offspring's reproductive health in the long term. The use of alpha-lipoic acid (ALA) as a dietary supplement during pregnancy has increased greatly in recent years and has been known to have positive effects on various pregnancy outcomes including miscarriage, diabetic embryopathy, preterm delivery, and congenital malformations. AimTo evaluate the effects of tobacco smoke exposure (TSE) on sexual behavior, reproductive parameters, and testicles in adult male rats and to reveal the possible role of ALA administration on these parameters. MethodsPregnant rats (n = 7 per group) were treated with tobacco smoke (TS), ALA (20 mg/kg), and TS + ALA for a total of 11 weeks. The following parameters were compared with 8 control rats: puberty parameters, sexual behavior; levels of serum gonadotropins and testosterone, total antioxidant status, and total oxidant status; the expression of the apoptotic protease-activating factor-1 and caspase 9 mRNA levels in the testis; and assessment of immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay of testis. Main Outcome MeasureSexual behavior, changes in puberty parameters, and hormonal and genetic alterations were the outcomes analyzed in this study. ResultsMaternal TSE caused a significant decrease in the number of intromissions compared to the control group. Similarly, ALA decreased erectile function in sexual behavior by decreasing the number of intromissions and intromission ratio in the ALA group compared to the control group. In addition, TSE and ALA treatment caused an impairment of some consummatory sexual behaviors. Also, in parallel with this inhibitory effect, the age of pubertal onset was significantly delayed in the TS + ALA group compared to other groups. Also, histopathological changes in testicular tissue, oxidative stress markers, apoptotic index, and mRNA levels of apoptosis-related genes increased in all treatment groups. Clinical ImplicationsThe use of ALA and/or tobacco products during pregnancy may adversely affect the reproductive health of male newborns in the long term. Strengths & LimitationsTo the best of our knowledge, this study is the first to show the effects of maternal ALA treatment and/or TSE on the sexual behavior and reproductive parameters in male rats; however, the study is based on an animal model, and the present findings partially reflect the characteristics of human sexual behavior. ConclusionMaternal TSE and/or ALA treatment may impair sexual behavior in adulthood in male rats because of testicular damage caused by oxidative stress during gonadal development.Yardimci A, Akkoc RF, Tektemur A, et al. Chronic Maternal Tobacco Smoke Exposure and/or Alpha-Lipoic Acid Treatment Causes Long-Term Deterioration of Testis and Sexual Behavior in Adult Male Rats. J Sex Med 2020;17:1835–1847.

Amelioration of elastase‐induced lung emphysema and reversal of pulmonary hypertension by pharmacological iNOS inhibition in mice

Chronic obstructive pulmonary disease, encompassing chronic airway obstruction and lung emphysema, is a major worldwide health problem and a severe socio-economic burden. Evidence previously provided by our group has shown that inhibition of inducible NOS (iNOS) prevents development of mild emphysema in a mouse model of chronic tobacco smoke exposure and can even trigger lung regeneration. Moreover, we could demonstrate that pulmonary hypertension is not only abolished in cigarette smoke-exposed iNOS-/- mice but also precedes emphysema development. Possible regenerative effects of pharmacological iNOS inhibition in more severe models of emphysema not dependent on tobacco smoke, however, are hitherto unknown. We have established a mouse model using a single dose of porcine pancreatic elastase or saline, intratracheally instilled in C57BL/6J mice. Emphysema, as well as pulmonary hypertension development was determined by both structural and functional measurements. Our data revealed that (i) emphysema is fully established after 21 days, with the same degree of emphysema after 21 and 28 days post instillation, (ii) emphysema is stable for at least 12 weeks and (iii) pulmonary hypertension is evident, in contrast to smoke models, only after emphysema development. Oral treatment with the iNOS inhibitor N(6)-(1-iminoethyl)-l-lysine (L-NIL) was started after emphysema establishment and continued for 12 weeks. This resulted in significant lung regeneration, evident in the improvement of emphysema and reversal of pulmonary hypertension. Our data indicate that iNOS is a potential new therapeutic target to treat severe emphysema and associated pulmonary hypertension. This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

Tobacco Smoke Extract Causes Mitochondrial Dysfunction Associated with Myotube Atrophy

INTRODUCTIONWhereas the link between chronic tobacco smoke (TS) exposure and development of disease (e.g., chronic obstructive pulmonary disease; COPD) is well established, how chronic TS exposure relates to the mitochondrial dysfunction and muscle atrophy seen in COPD remains unclear.METHODSWe exposed C2C12 myotubes to TS extract (TSE) on day 5 post‐differentiation to determine the impact of TS on mitochondrial function (respiration, reactive oxygen species [ROS] emission and sensitivity to mitochondrial permeability transition [MPT]), and myotube atrophy. Briefly, for respiration experiments myotubes were treated with 0.02% TSE or vehicle (DMSO) for 24 h, and subsequently washed and assessed for maximal uncoupled respiration in an oxygraph using carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; 250 nM – 1.5 μM). For ROS experiments, following 24 h TSE or DMSO treatment myotubes were incubated with the superoxide indicator MitoSox (500 nM) for 15 min and imaged for MitoSox positive area. For MPT experiments, after 24 h TSE or DMSO treatment myotubes were fixed in 4% PFA and incubated with anti‐Apoptosis Inducing Factor (AIF; a protein released from mitochondria during MPT) and NucBlue (to stain nuclei). Images (n=5 per well, 8 wells per treatment) were analysed using Image J to quantify the fraction of AIF co‐localizing with nuclei using the Manders coefficient. For myotube atrophy experiments, 24 h following TSE or DMSO treatment myotubes were fixed, labelled with anti‐sarcomeric myosin, and subsequently imaged to assess myotube area using automated batch processing to avoid human bias. Two‐tailed Students t‐tests were used to evaluate differences between DMSO vs. CSE with an alpha level of 0.05. Data are presented as mean±SD with exact p‐values.RESULTSTreatment of C2C12’s with TSE reduced maximal uncoupled respiration (TSE: 312.0±24.0 vs. DMSO: 415.1±45.5 pmoles/sec/μg, p=0.002) and increased MitoSox positive area (TSE: 8,607.0±2,963.1 vs. DMSO: 2,254.7±1,034.7 μm2, p=0.0001); the latter suggesting TSE increases ROS. AIF: nuclear co‐localization was greater in C2C12’s treated with TSE compared with vehicle (Manders coefficient in TSE: 0.66±0.08 vs. DMSO: 0.46±0.05, p=0.0001), suggesting TSE causes MPT. We also observed significantly smaller myotube area in TSE‐treated cells compared with vehicle (TSE: 62,972.9±43,527.5 vs. DMSO: 85,284.9±28,136.1 μm2, p=0.048), consistent with TS inducing myotube atrophy.DISCUSSIONOur experiments demonstrate that TSE exposure causes significant mitochondrial impairment, as evidenced by reduced respiratory capacity, increased ROS generation, and nuclear co‐localization with a mitochondrial protein released during MPT. Furthermore, TSE treatment caused significant myotube atrophy. These observations are consistent with chronic TS exposure playing an important role in the mitochondrial dysfunction and muscle atrophy seen with the TS‐related disease COPD.

Adult Life-Course Trajectories of Lung Function and the Development of Emphysema: The CARDIA Lung Study

BackgroundPeak lung function and rate of decline predict future airflow obstruction and nonrespiratory comorbid conditions. Associations between lung function trajectories and emphysema have not been explored. MethodsUsing data from the population-based CARDIA Study, we sought to describe the prevalence of visually ascertained emphysema at multiple time points and contextualize its development based upon participant’s adult life course measures of lung function. There were 3171 men and women enrolled at a mean age of 25 years, who underwent serial spirometric examinations through a mean age of 55 years. Trajectories for the change in percent-predicted forced expiratory volume in one second (FEV1) were determined by fitting a mixture model via maximum likelihood. Emphysema was visually identified on computed tomographic scans and its prevalence reported at mean ages of 40, 45, and 50 years. ResultsWe identified 5 trajectories describing peak and change in FEV1: “Preserved Ideal,” “Preserved Good,” “Preserved Impaired,” “Worsening,” and “Persistently Poor.” Ever smokers comprised part of all 5 trajectories. The prevalence of emphysema was 1.7% (n = 46; mean age of 40 years), 2.5% (n = 67; mean age of 45 years), and 7.1% (n = 189; mean age of 50 years). Of those with emphysema at a mean age of 50 years, 18.0% were never smokers. Worsening and poor lung health trajectories were associated with increased odds of future emphysema independent of chronic tobacco smoke exposure (odds ratio 5.06; confidence interval, 1.84-13.96; odds ratio 4.85; confidence interval, 1.43-16.44). ConclusionsLower peak and accelerated decline in FEV1 are risk factors for future emphysema independent of smoking status.

Conventional and electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In vivo evidence of a gender-specific cellular response to chronic cigarette smoke exposure

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) –driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid’s ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping.

Smoke-induced neuromuscular junction degeneration precedes the fibre type shift and atrophy in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is largely caused by smoking, and patient limb muscle exhibits a fast fibre shift and atrophy. We show that this fast fibre shift is associated with type grouping, suggesting recurring cycles of denervation-reinnervation underlie the type shift. Compared to patients with normal fat-free mass index (FFMI), patients with low FFMI exhibited an exacerbated fibre type shift, marked accumulation of very small persistently denervated muscle fibres, and a blunted denervation-responsive transcript profile, suggesting failed denervation precipitates muscle atrophy in patients with low FFMI. Sixteen weeks of passive tobacco smoke exposure in mice caused neuromuscular junction degeneration, consistent with a key role for smoke exposure in initiating denervation in COPD. A neurological basis for the fast fibre shift and atrophy seen in limb muscle of patients with chronic obstructive pulmonary disease (COPD) has not been considered previously. The objective of our study was: (1) to determine if denervation contributes to fast fibre shift and muscle atrophy in COPD; and (2) to assess using a preclinical smoking mouse model whether chronic tobacco smoke (TS) exposure could initiate denervation by causing neuromuscular junction (NMJ) degeneration. Vastus lateralis muscle biopsies were obtained from severe COPD patients [n=10 with low fat-free mass index (FFMI), 65years; n=15 normal FFMI, 65years) and healthy age- and activity-matched non-smoker control subjects (CON; n=11, 67years), to evaluate morphological and transcriptional markers of denervation. To evaluate the potential for chronic TS exposure to initiate these changes, we examined NMJ morphology in male adult mice following 16weeks of passive TS exposure. We observed a high proportion of grouped fast fibres and a denervation transcript profile in COPD patients, suggesting that motor unit remodelling drives the fast fibre type shift in COPD patient limb muscle. A further exacerbation of fast fibre grouping in patients with low FFMI, coupled with blunted reinnervation signals, accumulation of very small non-specific esterase hyperactive fibres and neural cell adhesion molecule-positive type I and type II fibres, suggests denervation-induced exhaustion of reinnervation contributes to muscle atrophy in COPD. Evidence from a smoking mouse model showed significant NMJ degeneration, suggesting that recurring denervation in COPD is probably caused by decades of chronic TS exposure.

A firsthand view of how secondhand smoke disrupts airway immunity

Chronic tobacco smoke exposure exacerbates Haemophilus influenzae infection by disrupting innate and adaptive immune responses in mice.

Association of Chronic Nicotine Inhalation with Hypertension in Mice

For decades it has been known that chronic tobacco smoke exposure is a risk factor for hypertension, predisposing to cardiovascular pathologies including stroke and heart failure. However, no prior study has evaluated the role of inhaled nicotine per se. Previous investigations found that subcutaneously‐administered nicotine acutely increases expression of angiotensin‐converting enzyme (ACE) and the angiotensin receptor (AT1R). These findings suggest RAS dysfunction may mediate nicotine‐induced hypertension through either increased expression of pro‐hypertensive proteins (ACE/AT1R) or suppressed expression of compensatory agents (ACE2/MasR).Accordingly, we hypothesized that chronic nicotine inhalation promotes hypertension by inducing RAS dysfunction within autonomic brain nuclei. To test this hypothesis we exposed male (n = 64) and female (n = 44) mice to air (control) or twelve hours of nicotine vapor nightly for eight weeks. Mean nocturnal systolic blood pressure (telemetry) increased between the baseline and final week of nicotine exposure (males: +19.71 ±0.65 mmHg, F = 528.2, n = 5, p &lt; 0.0001; females: +14.04 ±0.85 mmHg, F = 322.7, n = 4, p &lt; 0.0001). In females, a trend towards elevated urinary norepinephrine was observed (t = 1.751, p = 0.0993). In a pilot study, when we exposed rats for 3 weeks to 14 hours of nicotine nightly, the hypothalamic ACE2 mRNA levels were reduced (t = 2.490, p &lt; 0.05). In mice we found a trend towards increased expression of MasR mRNA in this brain region (t = 2.373, p = 0.0766).Together, these findings suggest that nicotine inhalation contributes to high blood pressure, possibly through enhanced sympathetic activity. Mechanistically, our pilot data indicate that impaired ACE2 expression occurs in tandem with a compensatory increase in MasR in the hypothalamus of subjects inhaling nicotine. Future studies will elucidate activity of additional RAS contributors in cell culture and clarify the nature of autonomic dysfunction in vivo (e.g., increased sympathetic output vs decreased vagal tone). Collectively, this project will provide crucial information regarding the risk of hypertension in users of nicotine inhalation devices (i.e. e‐cigarettes), improving the ability of clinicians to treat this condition.Support or Funding InformationThis work was supported by NIH NHLBI grants 1R01 HL135635 and 2R01 HL093178.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

8 and 16 weeks of Chronic Tobacco Smoke Exposure Negatively Impacts Peripheral Motor Axon and Neuromuscular Junction Morphology in the Diaphragm of Mice

IntroductionGlobally, chronic tobacco smoke (TS) exposure is the leading cause of preventable disease, and is the primary risk factor for the development of the top three causes of death: chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Importantly, TS‐related diseases are accompanied by similar skeletal muscle abnormalities, which contribute to poor outcomes and prognosis. Recent findings have implicated TS‐induced denervation in TS‐related locomotor muscle abnormalities. Given the important physiological role of the diaphragm, and that muscle abnormalities in tobacco smokers and people with TS‐related diseases manifest differently in the diaphragm than in locomotor muscle, the objective of this study was threefold: (1) to further assess the impact of TS exposure on the diaphragm; (2) to resolve whether the TS‐induced neuromuscular junction (NMJ) impact observed in locomotor muscle also occurs in the diaphragm; and (3) to investigate the progressive impact of TS exposure on morphological features of the motor axon and NMJ.Methods15‐wk‐old male C57Bl/6 wild‐type mice were randomly assigned to an 8‐ or 16‐wk period of either TS or control air exposure. Body mass was weighed at the beginning and end of the exposure periods. Immediately following sacrifice, epididymal fat, spleen, and liver were harvested and weighed in mice undergoing 16‐wk exposures. Diaphragm, gastrocnemius, plantaris, soleus, TA, and EDL muscles were harvested and weighed immediately following sacrifice in all mice. The diaphragm was used to analyze the morphology of motor axons and NMJs using immunofluorescent staining and confocal microscope imaging.Results8 and 16 weeks of TS exposure attenuated body growth (p &lt; 0.0005). Furthermore, 8 weeks (p &lt; 0.0001), but not 16 weeks, of TS exposure reduced muscle mass normalized to their respective controls in all muscles harvested. In the diaphragm, 8 and 16 weeks of TS exposure increased the fraction of NMJs lacking axonal input (p &lt; 0.05) or pre‐synaptic nerve terminal structures (p &lt; 0.005), the latter identified as denervated structures. Pre‐synaptic axon diameter was reduced following 16 (p &lt; 0.05), but not 8, weeks of TS exposure. 8 and 16 weeks of TS exposure led to reduced compactness of AChR structures (p &lt; 0.005), in the absence of AChR fragmentation.ConclusionsChronic TS exposure leads to reduced muscle mass of 8‐week TS‐exposed mice, as well as abnormal morphology of diaphragm motor axons and NMJs in 8‐ and 16‐wk TS‐exposed mice. Increased exposure length resulted in an exacerbation of the impact of TS on reduced pre‐synaptic axon growth and AChR compactness in 16‐wk versus 8‐wk TS exposed mice. These findings illustrate that chronic TS exposure results in specific morphological abnormalities in motor axons and NMJs in the diaphragm muscle.Support or Funding InformationGouvernement du Canada | Canadian Institutes of Health Research (Instituts de recherche en santé du Canada): Russell T. Hepple, 119583This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curcumin reversed chronic tobacco smoke exposure induced urocystic EMT and acquisition of cancer stem cells properties via Wnt/\u03b2-catenin

Tobacco smoke (TS) is the most important single risk factor for bladder cancer. Epithelial–mesenchymal transition (EMT) is a transdifferentiation process, involved in the initiation of TS-related cancer. Cancer stem cells (CSCs) have an essential role in the progression of many tumors including TS-related cancer. However, the molecular mechanisms of TS exposure induced urocystic EMT and acquisition of CSCs properties remains undefined. Wnt/β-catenin pathway is critical for EMT and the maintenance of CSCs. The aim of our present study was to investigate the role of Wnt/β-catenin pathway in chronic TS exposure induced urocystic EMT, stemness acquisition and the preventive effect of curcumin. Long time TS exposure induced EMT changes and the levels of CSCs’ markers were significant upregulated. Furthermore, we demonstrated that Wnt/β-catenin pathway modulated TS-triggered EMT and stemness, as evidenced by the findings that TS elevated Wnt/β-catenin activation, and that TS-mediated EMT and stemness were attenuated by Wnt/β-catenin inhibition. Treatment of curcumin reversed TS-elicited activation of Wnt/β-catenin, EMT and CSCs properties. Collectively, these data indicated the regulatory role of Wnt/β-catenin in TS-triggered urocystic EMT, acquisition of CSCs properties and the chemopreventive effect of curcumin.

Is periodontitis a comorbidity of COPD or can associations be explained by shared risk factors/behaviors?

COPD is recognized as having a series of comorbidities potentially related to common inflammatory processes. Periodontitis is one of the most common human inflammatory diseases and has previously been associated with COPD in numerous observational studies. As periodontitis and COPD are both chronic, progressive conditions characterized by neutrophilic inflammation with subsequent proteolytic destruction of connective tissue, it has been proposed that they share common pathophysiological processes. The mechanisms proposed to link COPD and periodontitis include mechanical aspiration of oral contents into the respiratory tree, overspill of locally produced inflammatory mediators into the systemic circulation or oral or lung-derived bacteremia activating an acute-phase response and also reactive oxygen species (ROS) and cytokine release by systemic neutrophils at distant sites. Studies of systemic neutrophils in COPD and chronic periodontitis describe altered cellular functions that would predispose to inflammation and tissue destruction both in the lung and in the mouth, again potentially connecting these conditions. However, COPD and periodontitis also share risk factors such as age, chronic tobacco smoke exposure, and social deprivation that are not always considered in observational and interventional studies. Furthermore, studies reporting associations have often utilized differing definitions of both COPD and periodontitis. This article reviews the current available evidence supporting the hypothesis that COPD and inflammatory periodontal disease (periodontitis) could be pathologically associated, including a review of shared inflammatory mechanisms. It highlights the potential limitations of previous studies, in particular, the lack of uniformly applied case definitions for both COPD and periodontitis and poor recognition of shared risk factors. Understanding associations between these conditions may inform why patients with COPD suffer such a burden of comorbid illness and new therapeutic strategies for both the diseases. However, further research is needed to clarify factors that may be directly causal as opposed to confounding relationships.

The Impact of Acute Smoking on Airway Gene-Expression

SESSION TITLE: Hot Topics in COPD SESSION TYPE: Original Investigation Slide PRESENTED ON: Tuesday, October 27, 2015 at 11:00 AM - 12:15 PM PURPOSE: We have previously reported gene expression changes in the bronchial airway epithelium in active smokers with longterm exposure to tobacco smoke. However, knowledge regarding the acute effects of smoking is still lacking. In this study we sought to describe the acute effects of smoking on airway epithelium gene expression. METHODS: Sixty-three individuals who are occasional light smokers without recent exposure to tobacco smoke (had not smoked a cigarette in at least 10 days) had airway epithelial cells collected from the mainstem bronchus by bronchoscopy, at baseline and at 24 hours after smoking three cigarettes. RNA isolated from the samples were processed and hybridized to Affymetrix Human Gene 1.0 ST microarrays. Gene expression differences between the baseline and post-smoking samples were assessed using a paired t-test. Enrichment of previously published smoking-related gene-expression signatures and biologic pathways were determined using Gene Set Enrichment Analysis (GSEA). RESULTS: We identified 80 genes which were differentially expressed between the baseline and post-smoking samples (FDR < 0.25). Forty-eight of the genes were induced by smoking and were related to cell survival, anti-apoptosis, xenobiotic metabolism, oxidant response, and innate immunity. Furthermore, we found that genes that change with acute exposure to tobacco smoke were enriched among genes that we previously identified as changing among chronic active smokers (GSEA FDR < 0.05). CONCLUSIONS: Acute exposure to as little as three cigarettes induces gene-expression alterations in the bronchial airway epithelium that resemble some of the changes seen in the airway of chronic active smokers. CLINICAL IMPLICATIONS: Understanding the similarity and differences between the biological effects of acute and chronic tobacco smoke exposure may have implications for understanding the pathogenesis of diseases associated with chronic tobacco exposure such as COPD and lung cancer. DISCLOSURE: The following authors have nothing to disclose: Ehab Billatos, Yaron Gesthalter, Nick ten Hacken, Dirkje Postma, Irene Heijink, Wim Timens, Corry-Anke Brandsma, Alen Faiz, Maarten Berge, Marc Lenburg, Avrum Spira No Product/Research Disclosure Information

Effect of full flavor and denicotinized cigarettes exposure on the brain microvascular endothelium: a microarray-based gene expression study using a human immortalized BBB endothelial cell line.

BackgroundTobacco smoke (TS) toxicity to the brain microvasculature is still an understudied area till date. NF-E2 related factor (Nrf2) is a key transcription factor responsible for activating the antioxidant response element (ARE) genes following an oxidative insult. Till date, several studies targeting the blood brain barrier (BBB) have shown some protective role of Nrf2 in ischemia–reperfusion (IR) injury, however, its functional role in chronic smokers subjected to a life-long oxidative stress has never been addressed. This is of crucial importance since smokers have a much higher risk for cerebrovascular stroke and tobacco smoke exposure has been clearly shown to enhance BBB damage following an ischemia/reperfusion injury. Thus, the goal of our study was to investigate the defense pathways activated at the BBB endothelial level by TS exposure. Specifically we focused on Nrf2 and nuclear factor kappa-light-chain-enhancer of activated B signaling response (NF-κβ) as the central protective mechanisms related to oxidative insult.ResultsWith the exception of Nicotine, both full flavor (3R4F) and decotinized (ULN) cigarettes activated Nrf2 and NFκβ pathways in hCMEC/D3 endothelial cells. Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11). Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced. Increase of P-gp functional activity and depletion of GSH were also observed. Strikingly, toxicity of denicotinized (“reduced exposure”) cigarettes was equivalent to 3R4F (or worse).ConclusionsThis study provides a detailed analysis of Nrf2-related cytoprotective mechanisms activated in response to 3R4F and ULN-derived TS exposure correlating the results with their oxidative and inflammatory potential. Toxicants present in soluble cigarette smoke extracts (CSE) and not nicotine seem to be the primary determinant of vascular toxicity. In this respect our results from this and previous studies suggest that chronic TS exposure can overcome Nrf2 and NFκB-p65 dependent cytoprotective mechanisms of the brain microvascular endothelium possibly leading to BBB impairment and loss of BBB integrity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-015-0173-3) contains supplementary material, which is available to authorized users.