Weeks Of Cigarette Smoke Exposure Research Articles

Modified Bushen Yiqi Formula mitigates pulmonary inflammation and airway remodeling by inhibiting neutrophils chemotaxis and IL17 signaling pathway in rats with COPD

Ethnopharmacological relevanceChronic obstructive pulmonary disease (COPD) is a major global health concern characterized by pulmonary inflammation and airway remodeling. Traditional Chinese medicine, such as Modified Jiawei Bushen Yiqi Formula (MBYF), has been used as a complementary therapy for COPD in China. Aim of the studyTo investigate the therapeutic potential of MBYF in a rat model of COPD induced by cigarette smoke (CS) exposure and explore the underlying mechanism. Materials and methodsThe COPD rat model was established through 24 weeks of CS exposure, with MBYF administration starting in the 9th week. Pulmonary function, histological analysis, inflammatory cell count and molecular assays were employed to assess the effects of MBYF on airway remodeling, pulmonary inflammation, neutrophils chemotaxis and the IL17 signaling pathway. ResultsMBYF treatment effectively delayed airway remodeling, as evidenced by improved pulmonary function parameters. Histological examination and bronchoalveolar lavage fluid analysis revealed that MBYF mitigated CS-induced pulmonary inflammation by reducing inflammatory cell infiltration. Pharmacological network analysis suggested that MBYF may act through the IL17 signaling pathway to regulate inflammatory responses. RNA-sequencing and molecular assays indicated that MBYF inhibited neutrophils chemotaxis through downregulating the CXCL1/CXCL5/CXCL8-CXCR2 axis, and suppressed IL17A, IL17F and its downstream cytokines, including IL6, TNFα, IL1β, and COX2. Furthermore, MBYF inhibited the activation of NF-κB and MAPKs in the IL17 signaling pathway. ConclusionMBYF exhibits potential as an adjunct or alternative treatment for COPD, effectively mitigating CS-induced pulmonary inflammation and airway remodeling through the inhibition of neutrophil chemotaxis and IL17 signaling pathway.

Cigarette smoke exposure reduces hemorrhagic shock induced circulatory dysfunction in mice with attenuated glucocorticoid receptor function.

IntroductionWe previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS.MethodsAfter 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid–base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range.ResultsNor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals.ConclusionWhether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

Interaction between cigarette smoke exposure and physical training on inflammatory and oxidative profile in mice muscle

Regular physical training and cigarette smoke exposure (CSE) have opposite effects on physical performance, antioxidant, and inflammatory profile. However, the interaction between these events is not well studied. We aimed to investigate how regular physical training and CSE interact, and in what is the outcome of this interaction on the physical performance, skeletal muscle antioxidant defense and molecular profile response of pro and anti-inflammatory cytokines. Male C57BL/6 mice were randomly divided into 4 groups (n = 8/group): 1) Sedentary group (SED); 2) 4 weeks of control, followed by 4 weeks of CSE (SED + CSEG); 3) Physically active (PA) along 8 weeks (forced swim training, 5 times a week); 4) Physically active and exposed to the cigarette smoke (PA + CSEG), group submitted to forced swim training for 4 weeks, followed by 4 weeks of concomitant training and CSE. Physical performance was evaluated before and after the experimental period (8 weeks), total peroxidase and glutathione peroxidase (GPx) activities, expression of genes encoding TNF-α, MCP-1, IL1β, IL-6, IL-10, TGF-β, HO-1 and the TNF-α/IL-10 ratio were determined from gastrocnemius muscle at the end of experimental period. The CSE attenuated the aerobic capacity adaptation (time to exhaustion in swimming forced test) promoted by physical training and inhibit the improvement in local muscle resistance (inverted screen test). The regular physical training enhanced the antioxidant defense, but the CSE abrogated this benefit. The CSE induced a harmful pro-inflammatory profile in skeletal muscle from sedentary animals whereas the regular physical training induced an opposite adaptation. Likewise, the CSE abolished the protective effect of physical training. Together, these results suggest a negative effect of CSE including, at least in part, the inhibition/attenuation of beneficial adaptations from regular physical training.

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Apocynin prevents cigarette smoking-induced loss of skeletal muscle mass and function in mice by preserving proteostatic signalling.

Background and PurposeSkeletal muscle dysfunction is a major comorbidity of chronic obstructive pulmonary disease (COPD). This type of muscle dysfunction may be a direct consequence of oxidative insults evoked by cigarette smoke (CS) exposure. The present study examined the effects of a potent Nox inhibitor and reactive oxygen species (ROS) scavenger, apocynin, on CS‐induced muscle dysfunction.Experimental ApproachMale BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks, with or without the coadministration of apocynin (5 mg·kg−1, i.p.). C2C12 myotubes exposed to either hydrogen peroxide (H2O2) or water‐soluble cigarette smoke extract (CSE) with or without apocynin (500 nM) were used as an experimental model in vitro.Key ResultsEight weeks of CS exposure caused muscle dysfunction in mice, reflected by 10% loss of muscle mass and 54% loss of strength of tibialis anterior which were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2O2 or CSE caused myofibre wasting, accompanied by ~50% loss of muscle‐derived insulin‐like growth factor (IGF)‐1 and two‐fold induction of Cybb, independent of cellular inflammation. Expression of myostatin and MAFbx, negative regulators of muscle mass, were up‐regulated under H2O2 but not CSE conditions. Apocynin treatment abolished CSE‐induced Cybb expression, preserving muscle‐derived IGF‐1 expression and signalling pathway downstream of mammalian target of rapamycin (mTOR), thereby preventing myofibre wasting.Conclusion and ImplicationsTargeted pharmacological inhibition of Nox‐derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.

Abstract A06: Impact of cigarette smoke exposure on acute myeloid leukemia progression

Abstract Cigarette smoke is known to cause lung cancer but is also associated with increased incidence of leukemia. Acute myeloid leukemia (AML) is the most lethal form of leukemia, and patients who are or were smokers have increased risk of developing AML and have worse prognosis. There is also an increased incidence of AML in children whose parents smoked either during or after the pregnancy. It is currently unknown how cigarette smoke exposure (CSE) may be impacting leukemic progression or treatment efficacy in AML. Cigarette smoking increases levels of reactive oxygen species (ROS) in the tissue of smokers, including peripheral blood samples. Oxidative stress can alter epigenetic signaling, including DNA methylation. Oxidation of methylated DNA and oxidative lesions on DNA can alter normal DNA methylation patterns. Smokers have altered DNA methylation that can be retained decades after the cessation of the habit, and these changes can be found in the blood cells of smokers. Cigarette smoke condensate (CSC) treatment has also been seen to epigenetically prime nontransformed human bronchial cells for malignant transformation. These occurrences have been seen in the context of lung cancer and lung damage, but studies into other tissue damage have largely been overlooked. Thus, we hypothesize that CSE impacts leukemic progression and treatment in AML through these mechanisms. We have developed a model to study the impact of CSE on AML in mice. NOD-SCID mice are smoke exposed for two weeks prior to being engrafted with luciferase-labeled human AML cells. The CSE is performed in the SCIREQ Cigarette Smoking Robot, with 3R4F research cigarettes 5 days/week. We include experimental arms of smoking cessation (who stop once evidence of leukemic engraftment is detected) and continued smoking, to model patients who either stop or continue smoking upon diagnosis of AML. Leukemic burden is quantified through bioluminescent imaging throughout the experiment. From these models, we have observed that two weeks of CSE is sufficient to significantly increase (p-value<0.0001) early leukemic burden as compared to non-smoke exposed mice, in FLT3-ITD AML-bearing mice. Spleen samples revealed trends of increased levels of superoxides in the CSE mice. Currently, immunohistochemistry is being performed on collected organs to detect oxidative stress markers. Additionally, mouse spleen DNA underwent reduced representative bisulfide sequencing; the levels of DNA methylation in the mouse host cells and in the leukemia are compared between our CSE groups. This is the first description of CSE promoting leukemia growth in mouse model, which will enable the study of mechanistic aspects. This may help define if unique treatment strategies are needed for these patients. Citation Format: Yue Lu, Marcos Estecio, Marina Konopleva, Elias Jabbour, Joya Chandra, Mary Figueroa. Impact of cigarette smoke exposure on acute myeloid leukemia progression [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A06.

Cigarette smoke-initiated autoimmunity facilitates sensitisation to elastin-induced COPD-like pathologies in mice.

It is currently not understood whether cigarette smoke exposure facilitates sensitisation to self-antigens and whether ensuing auto-reactive T cells drive chronic obstructive pulmonary disease (COPD)-associated pathologies.To address this question, mice were exposed to cigarette smoke for 2 weeks. Following a 2-week period of rest, mice were challenged intratracheally with elastin for 3 days or 1 month. Rag1-/- , Mmp12-/- , and Il17a-/- mice and neutralising antibodies against active elastin fragments were used for mechanistic investigations. Human GVAPGVGVAPGV/HLA-A*02:01 tetramer was synthesised to assess the presence of elastin-specific T cells in patients with COPD.We observed that 2 weeks of cigarette smoke exposure induced an elastin-specific T cell response that led to neutrophilic airway inflammation and mucus hyperproduction following elastin recall challenge. Repeated elastin challenge for 1 month resulted in airway remodelling, lung function decline and airspace enlargement. Elastin-specific T cell recall responses were dose dependent and memory lasted for over 6 months. Adoptive T cell transfer and studies in T cells deficient Rag1-/- mice conclusively implicated T cells in these processes. Mechanistically, cigarette smoke exposure-induced elastin-specific T cell responses were matrix metalloproteinase (MMP)12-dependent, while the ensuing immune inflammatory processes were interleukin 17A-driven. Anti-elastin antibodies and T cells specific for elastin peptides were increased in patients with COPD.These data demonstrate that MMP12-generated elastin fragments serve as a self-antigen and drive the cigarette smoke-induced autoimmune processes in mice that result in a bronchitis-like phenotype and airspace enlargement. The study provides proof of concept of cigarette smoke-induced autoimmune processes and may serve as a novel mouse model of COPD.

Serotonin receptors 5-HTR2A and 5-HTR2B are involved in cigarette smoke-induced airway inflammation, mucus hypersecretion and airway remodeling in mice

BackgroundCigarette smoke plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Recently, elevated serotonin (5-HT) levels were found in the plasma of COPD patients. The role of 5-HT and its receptors in airway inflammation and remodeling induced by cigarette smoke is unclear. MethodsBALB/c mice received the 5-HTR2A inhibitor ketanserin, the 5-HTR2B inhibitor RS-127445 or the natural 5-HTR2A/2B inhibitor quercetin intraperitoneally, then were exposed to cigarette smoke for 6 or 12 weeks. Control mice received placebo and were exposed to room air or cigarette smoke. Mice were sacrificed and bronchial alveolar lavage fluid (BALF) and lung tissue samples were collected. ResultsImmunohistochemistry and western blot confirmed an increase in both 5-HTR2A and 5-HTR2B expression in mouse lungs after exposure to cigarette smoke for 6 and 12 weeks. Cigarette smoke induced accumulation of macrophages and neutrophils and increased levels of inflammatory cytokines, including IL-1β and TNF-ɑ, in BALF and lung tissue; these effects were inhibited by ketanserin, RS-127445 and quercetin. Pretreatment with 5-HT receptor antagonists suppressed the goblet cell hyperplasia induced by 6- or 12-week exposure to cigarette smoke, based on Alcian blue-periodic acid Schiff staining. After 12 weeks of cigarette smoke exposure, Masson's staining showed fibrosis surrounding the mouse airways, and inhibitor pretreatment significantly attenuated the thickening and collagen deposition around the small airways. ConclusionsOur results suggest that cigarette smoke-induced airway inflammation and small airway remodeling are partially mediated by 5-HTR2A and 5-HTR2B, which could be a new therapeutic target for airway remodeling in COPD.

In-Depth Characterization of the Effects of Cigarette Smoke Exposure on the Acute Trauma Response and Hemorrhage in Mice.

Hemorrhagic shock accounts for a large amount of trauma-related mortality. The severity of trauma can be further aggravated by an additional blunt chest trauma (TxT), which independently contributes to mortality upon the development of an acute lung injury (ALI). Besides, cigarette smoke (CS) exposure before TxT enhanced posttraumatic inflammation, thereby aggravating ALI. We therefore aimed to characterize the impact of an acute and/or chronic lung injury on organ dysfunction in a murine model of traumatic hemorrhagic shock (HS). After 3 weeks of CS exposure, anesthetized mice underwent HS with/without TxT. Hemorrhagic shock was implemented for 1 h followed by retransfusion of shed blood and intensive care therapy for 4 h including lung-protective mechanical ventilation, fluid resuscitation, and noradrenaline titrated to maintain mean arterial pressure ≥50 mmHg. Lung mechanics and gas exchange were assessed together with systemic hemodynamics, metabolism, and acid-base status. Postmortem blood and tissue samples were analyzed for cytokine and chemokine levels, protein expression, mitochondrial respiration, and histological changes. CS exposure and HS alone coincided with increased inflammation, decreased whole blood sulfide concentrations, and decreased diaphragmatic mitochondrial respiration. CS-exposed mice, which were subjected to TxT and subsequent HS, showed hemodynamic instability, acute kidney injury, and high mortality. Chronic CS exposure per se had the strongest impact on inflammatory responses. The degree of inflammation was similar upon an additional TxT, however, mice presented with organ dysfunction and increased mortality rates. Hence, in mice the degree of inflammation may be dissociated from the severity of organ dysfunction or injury.

Effects of cigarette smoke and tobacco heating aerosol on color stability of dental enamel, dentin, and composite resin restorations.

To test if cigarette smoke (CS) causes discoloration of enamel, dentin, and composite resin restorations and induces color mismatch between dental hard tissues and the restorations, and to compare the findings with the effects of aerosol generated by the tobacco heating system (THS) 2.2. Twenty-two human premolars were prepared with Class V cavities restored with Filtek Supreme Ultra (3M Espe) composite resin. Teeth were divided into two groups and exposed to either CS from 20 reference cigarettes (3R4F) or aerosol from 20 THS 2.2 tobacco heat sticks 4 days a week for 3 weeks. CIE L*a*b* color was assessed before and after exposure and brushing at 1, 2, and 3 weeks. Color match, marginal discoloration, marginal integrity, and surface texture of the Class V restoration were assessed according to a modified US Public Health Service (USPHS) criterion. Marked discoloration of enamel and dentin was observed following 3 weeks of CS exposure (ΔE = 8.8 ± 2.6 and 21.3 ± 4.4, respectively), and color mismatch occurred between the composite resin restorations (ΔE = 25.6 ± 3.8) and dental hard tissues. Discoloration was minimal in the enamel, dentin, and composite resin restorations in the THS 2.2 group, and no color mismatch was observed after 3 weeks of THS 2.2 aerosol exposure. CS causes significant tooth discoloration and induces color mismatch between dental hard tissues and composite resin restorations. Reducing or eliminating the deposits derived from tobacco combustion could minimize the impact of tobacco products on tooth discoloration.

Interaction of the hydrogen sulfide system with the oxytocin system in the injured mouse heart

BackgroundBoth the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) and oxytocin/oxytocin receptor (OT/OTR) systems have been reported to be cardioprotective. H2S can stimulate OT release, thereby affecting blood volume and pressure regulation. Systemic hyper-inflammation after blunt chest trauma is enhanced in cigarette smoke (CS)-exposed CSE−/− mice compared to wildtype (WT). CS increases myometrial OTR expression, but to this point, no data are available on the effects CS exposure on the cardiac OT/OTR system. Since a contusion of the thorax (Txt) can cause myocardial injury, the aim of this post hoc study was to investigate the effects of CSE−/− and exogenous administration of GYY4137 (a slow release H2S releasing compound) on OTR expression in the heart, after acute on chronic disease, of CS exposed mice undergoing Txt.MethodsThis study is a post hoc analysis of material obtained in wild type (WT) homozygous CSE−/− mice after 2-3 weeks of CS exposure and subsequent anesthesia, blast wave-induced TxT, and surgical instrumentation for mechanical ventilation (MV) and hemodynamic monitoring. CSE−/− animals received a 50 μg/g GYY4137-bolus after TxT. After 4h of MV, animals were exsanguinated and organs were harvested. The heart was cut transversally, formalin-fixed, and paraffin-embedded. Immunohistochemistry for OTR, arginine-vasopressin-receptor (AVPR), and vascular endothelial growth factor (VEGF) was performed with naïve animals as native controls.ResultsCSE−/− was associated with hypertension and lower blood glucose levels, partially and significantly restored by GYY4137 treatment, respectively. Myocardial OTR expression was reduced upon injury, and this was aggravated in CSE−/−. Exogenous H2S administration restored myocardial protein expression to WT levels.ConclusionsThis study suggests that cardiac CSE regulates cardiac OTR expression, and this effect might play a role in the regulation of cardiovascular function.

MicroRNA profiling in lung tissue and bronchoalveolar lavage of cigarette smoke-exposed mice and in COPD patients: a translational approach

Chronic obstructive pulmonary disease (COPD) is characterized by a progressive airflow limitation and is associated with a chronic inflammatory response in both airways and lungs. microRNAs (miRNAs) are often highly conserved between species and have an intricate role within homeostatic conditions and immune responses. Also, miRNAs are dysregulated in smoking-associated diseases. We investigated the miRNA profile of 523 miRNAs by stem-loop RT-qPCR in lung tissue and cell-free bronchoalveolar lavage (BAL) supernatant of mice exposed to air or cigarette smoke (CS) for 4 or 24 weeks. After 24 weeks of CS exposure, 31 miRNAs were differentially expressed in lung tissue and 78 in BAL supernatant. Next, we correlated the miRNA profiling data to inflammation in BAL and lung, obtained by flow cytometry or ELISA. In addition, we surveyed for overlap with newly assessed miRNA profiles in bronchial biopsies and with previously assessed miRNA profiles in lung tissue and induced sputum supernatant of smokers with COPD. Several miRNAs showed concordant differential expression between both species including miR-31*, miR-155, miR-218 and let-7c. Thus, investigating miRNA profiling data in different compartments and both species provided accumulating insights in miRNAs that may be relevant in CS-induced inflammation and the pathogenesis of COPD.

Cigarette smoke attenuated colon inflammation through the induction of regulatory T cells in ulcerative colitis model

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory disease induced by dysregulation of immune response in intestinal mucosa. Several cohort studies have shown that cigarette smoke (CS) is the protective factor for UC, but the underlying mechanisms are not fully elucidated. Here, we evaluated the influence of CS in the experimental UC model. Methods UC was induced by dextran sodium sulfate (DSS) during three weeks of CS exposure, and mice were sacrificed at day 7 following UC induction. Colon pathology, lymphocytes recruitment into intestinal mucosa, and gut microbiota were analyzed. Results CS exposure suppressed colon inflammation and disease activity in DSS-induced colitis model. CS exposed mice displayed significant reduction of activated CD8+ T cells in colon lamina propria, whereas regulatory T cells (Tregs) were significantly increased. In addition, Ruminococcus, which belongs to Clostridium cluster IV, increased in fecal microbiota of CS exposed mice, suggesting that Tregs are induced by CS exposure through the alteration of microbiota composition. Conclusions CS might suppress colon inflammation in UC through the alteration of microbiota composition and immune system including Tregs.

The Role of Cystathionine-γ-Lyase In Blunt Chest Trauma in Cigarette Smoke Exposed Mice.

Pretraumatic cigarette smoke (CS) exposure aggravates posttraumatic acute lung injury (ALI). Cystathionine-γ-lyase (CSE) protects against ALI and CS exposure-induced chronic obstructive lung disease (COPD). Therefore, we tested the hypothesis whether genetic CSE knockout (CSE) would aggravate posttraumatic ALI after CS exposure. After 3 to 4 weeks of CS exposure, anesthetized wild-type (WT) and CSE mice underwent blunt chest trauma, surgical instrumentation and 4 h of lung-protective mechanical ventilation. We measured hemodynamics, lung mechanics, gas exchange, metabolism, and acid-base status together with blood and tissue cytokine and chemokine levels, tissue expression of mediator proteins, parameters of oxidative and nitrosative stress, and histology. CSE mice without CS exposure showed higher cytokine and chemokine levels, and this was further enhanced by CS exposure, particularly in males. CS exposure in WT mice aggravated posttraumatic alveolar membrane thickening, dystelectasis, and inflammatory cell accumulation, which was associated with higher thoracopulmonary compliance. Pretraumatic CS exposure in CSE mice produced a similar response, except for less alveolar membrane thickening, most likely due to lung hyperinflation. CS-exposed WT mice showed the most pronounced metabolic acidosis, while CS exposure in CSE mice resulted in the lowest blood glucose levels. Urinary output and anesthesia rate were highest in male CS-exposed CSE animals. In conclusion, in murine acute-on-chronic pulmonary disease, CSE knockout aggravated posttraumatic inflammation, which was further worsened upon pretraumatic CS exposure, and this effect was particularly pronounced in males. Hence, maintaining CSE expression is critically important for stress adaptation during ALI and CS-induced COPD, most likely in a gender-dependent manner.

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

Effect of Simvastatin on 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension

Background/Aims: To investigaterole of serotonin (5-HT) and serotonin transporter (5-HTT) in a rat model of cigarette smoke-induced pulmonary artery hypertension (PAH) and the effect of statins on regulating 5HT and 5-HTT. Methods: A rat model of COPD comorbid with PAH was established by cigarette smoke exposure with or without simvastatin administration. The smoking and the simvastatin plus smoking groups were exposed to cigarette smoke daily, and the latter received simvastatin at 5mg/kg, once a day. After 16 weeks of cigarette smoke exposure, body weight and mean pulmonary arterial pressure (mPAP) were measured, bronchoalveolar lavage (BAL) was performed, and lung tissues and blood samples were collected to determine cardiopulmonary pathology, physiological indices, blood levelof 5-HT and expression of 5-HTT in the lung. Results: In addition to alveolar structural damage (COPD-like injury), chronic cigarette smoke exposure lead to pulmonary artery remodeling and PAH as evidenced by significant elevation of mPAP, RVHI, WT%and WA%. Cigarette smoke exposure resulted in significant reduction in animal body weight, and simvastatin significantly prevented smoke-induced weight loss. The number of inflammatory cells in BALF was dramatically increased in smoke exposed rats, and simvastatin dampened the number of leukocytes, neutrophils, lymphocytes, and macrophages. In addition, circulating 5-HTand expression of 5-HTT in the lung were significantly increased in the smoked rats compared to control rats, and it was significantly reduced by simvastatin. Alteration of BALF inflammatory cells, 5-HT and 5-HTT was significantly correlated with changes of mPAP, RVHI, WT% and WA%. Conclusions: Cigarette smoke exposure could result in not only COPD, but also PAH, which may attribute to the alteration of blood 5-HT and lung tissue 5-HTT. Simvastatin could significantly inhibited 5-HT and 5-HTT expression, and by which mechanism, it may protect animals from development of PAH.

Cigarette smoke-induced lung endothelial apoptosis and emphysema are associated with impairment of FAK and eIF2α

Lung endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. However, the mechanism underlying cigarette smoke (CS)-induced lung EC apoptosis and emphysema is not well defined. We have previously shown that cigarette smoke extract (CSE) decreased focal adhesion kinase (FAK) activity via oxidative stress in cultured lung EC. In this study, we compared FAK activation in the lungs of highly susceptible AKR mice and mildly susceptible C57BL/6 mice after exposure to CS for three weeks. We found that three weeks of CS exposure caused mild emphysema and increased lung EC apoptosis in AKR mice (room air: 12.8±5.6%; CS: 30.7±3.7%), but not in C57BL/6 mice (room air: 0±0%; CS: 3.5±1.7%). Correlated with increased lung EC apoptosis and early onset of emphysema, FAK activity was reduced in the lungs of AKR mice, but not of C57BL/6 mice. Additionally, inhibition of FAK caused lung EC apoptosis, whereas over-expression of FAK prevented CSE-induced lung EC apoptosis. These results suggest that FAK inhibition may contribute to CS-induced lung EC apoptosis and emphysema. Unfolded protein response (UPR) and autophagy have been shown to be activated by CS exposure in lung epithelial cells. In this study, we noted that CSE activated UPR and autophagy in cultured lung EC, as indicated by enhanced eIF2α phosphorylation and elevated levels of GRP78 and LC3B-II. However, eIF2α phosphorylation was significantly reduced by three-weeks of CS exposure in the lungs of AKR mice, but not of C57BL/6 mice. Markers for autophagy activation were not significantly altered in the lungs of either AKR or C57BL/6 mice. These results suggest that CS-induced impairment of eIF2α signaling may increase the susceptibility to lung EC apoptosis and emphysema. Taken together, our data suggest that inhibition of eIF2α and FAK signaling may play an important role in CS-induced lung EC apoptosis and emphysema.

The Role of Soluble Guanylyl Cyclase in Chronic Obstructive Pulmonary Disease

Soluble guanylyl cyclase (sGC), a cyclic guanosine 5'-monophosphate-generating enzyme, regulates smooth muscle tone and exerts antiinflammatory effects in animal models of asthma and acute lung injury. In chronic obstructive pulmonary disease (COPD), primarily caused by cigarette smoke (CS), lung inflammation persists and smooth muscle tone remains elevated, despite ample amounts of nitric oxide that could activate sGC. To determine the expression and function of sGC in patients with COPD and in a murine model of COPD. Expression of sGCα1, α2, and β1 subunits was examined in lungs of never-smokers, smokers without airflow limitation, and patients with COPD; and in C57BL/6 mice after 3 days, 4 weeks, and 24 weeks of CS exposure. The functional role of sGC was investigated in vivo by measuring bronchial responsiveness to serotonin in mice using genetic and pharmacologic approaches. Pulmonary expression of sGC, both at mRNA and protein level, was decreased in smokers without airflow limitation and in patients with COPD, and correlated with disease severity (FEV1%). In mice, exposure to CS reduced sGC, cyclic guanosine 5'-monophosphate levels, and protein kinase G activity. sGCα1(-/-) mice exposed to CS exhibited bronchial hyperresponsiveness to serotonin. Activation of sGC by BAY 58-2667 restored the sGC signaling and attenuated bronchial hyperresponsiveness in CS-exposed mice. Down-regulation of sGC because of CS exposure might contribute to airflow limitation in COPD.

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

BackgroundSkeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.MethodsA mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.ResultsGastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.ConclusionsRepeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.

Comparison of Biological Responses in Rats Under Various Cigarette Smoke Exposure Conditions

A variety of exposure regimens of cigarette smoke have been used in animal models of lung diseases. In this study, we compared biological responses of smoke exposure in rats, using different smoke concentrations (wet total particulate matter [WTPM]), daily exposure durations, and total days of exposure. As a range-finding acute study, we first compared pulmonary responses between SD and F344 strains after a single nose-only exposure to mainstream cigarette smoke or LPS. Secondly, F344 rats were exposed to cigarette smoke for 2 or 13 weeks under the comparable daily exposure dose (WTPM concentration x daily exposure duration; according to Haber’s rule) but at a different WTPM concentration or daily exposure duration. Blood carboxylhemoglobin was increased linearly to the WTPM concentration, while urinary nicotine plus cotinine value was higher for the longer daily exposure than the corresponding shorter exposure groups. Gamma glutamyl transferase activity in bronchoalveolar lavage fluid (BALF) was increased dose dependently after 2 and 13 weeks of cigarette smoke exposure, while the neutrophil content in BALF was not increased notably. Smoke-exposed groups showed reduced body weight gain and increased relative lung and heart weights. While BALF parameters and the relative lung weights suggest pulmonary responses, histopathological examination showed epithelial lesions mainly in the upper respiratory organs (nose and larynx). Collectively, the results indicate that, under the employed study design, the equivalent daily exposure dose (exposure concentration x duration) induces equivalent pulmonary responses in rats.