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Electronic cigarettes (e-cigs) are advertised as a less harmful nicotine delivery system or as a new smoking cessation tool. We aimed to assess the in vivo effects of e-cig vapor in the lung and to compare them to those of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene glycol/vegetable glycerol (PG:VG-Sol; 1:1), 2) PG:VG with nicotine (G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N+F) and determined oxidative stress, inflammation, and pulmonary mechanics. E-cig vapors, especially PG:VG-N+F, increased bronchoalveolar lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF and lung oxidative stress markers at least comparably and in many cases more than CS. BALF protein content at both time points studied was only elevated in the PG:VG-N+F group. After 3 days, PG:VG-Sol altered tissue elasticity, static compliance, and airway resistance, whereas after 4 wk CS was the only treatment adversely affecting these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N+F groups. Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics. In many cases, the added flavor in e-cigs exacerbated the detrimental effects of e-cig vapor. We conclude that both e-cig vaping and conventional cigarette smoking negatively impact lung biology.

Nitric oxide (NO) is a gaseotransmitter, which is involved in many signaling processes in health and disease. Three enzymes generate NO from L-arginine, with citrulline formed as a by-product: neuronal NO synthase (nNOS or NOS1), endothelial NOS (eNOS or NOS3) and inducible NOS (iNOS or NOS2). NO is a ligand of soluble guanylyl cyclase (sGC), an intracellular heterodimer enzyme that catalyzes the con- version of guanosine triphosphate (GTP) to cyclic GMP (cGMP). cGMP further activates protein kinase G that eventually reduces the smooth muscle tone in bronchi or vessels. Phosphodiesterase 5 (PDE5) de- grades cGMP to GMP. However, NO reacts with superoxide anion (O � ), leading to formation of the pro- inflammatory molecule peroxynitrite. Under physiological conditions, NO plays a homeostatic bronchoprotective role in healthy subjects. In obstructive airway diseases, NO can be beneficial by its bronchodilating effect, but could also be detrimental by the formation of peroxynitrite. Since asthma and COPD are associated with increased levels of exhaled NO, chronic inflammation and increased airway smooth muscle tone, the NO/sGC/cGMP pathway could be involved in these highly prevalent obstructive airway diseases. Here we review the involvement of NO, NO synthases, guanylyl cyclases, cGMP and phophodiesterase-5 in asthma and COPD and potential therapeutic approaches to modulate this pathway.

Introduction: Resistive breathing (RB) is the hallmark of diseases of airway obstruction, such as chronic obstructive pulmonary disease (COPD), especially during exacerbations. RB results in increased mechanical stress and inflammation in the lung of previously healthy mice. Cigarette smoke (CS) exposure, the main cause of COPD, also induces pulmonary inflammation. Aim: To investigate the potential synergistic effect of the combination of RB with CS exposure model. Methods: Mice were exposed to CS of 5 reference cigarettes, 4 times daily, 5 days per week for one month. Control mice were exposed to room air. By the end of the month mice were subjected to RB for 24 hours by tracheal banding or were sham operated. Tracheal banding was induced by placing the mice under a surgical microscope, the trachea was exposed and a nylon band was placed around the trachea and firmly sutured to provide a surface area 50% of the initial. Following 24 hours, respiratory system mechanics were assessed by the forced oscillation technique. Bronchoalveolar lavage (BAL) was performed and total and differential cell count and total protein level were measured in BAL fluid. Results: CS exposure was associated with increased BAL cellularity (10-fold to ctr, p Conclusions: Combining resistive breathing with cigarette smoke exposure results into augmented inflammatory responses.

Konstantinos Loverdos,1 Dimitrios Toumpanakis,1 Eleni Litsiou,1 Vassiliki Karavana,1 Constantinos Glynos,1 Christina Magkou,2 Stamatios Theocharis,3 Theodoros Vassilakopoulos1 1Department of Critical Care, Pulmonary Unit and Marianthi Simou Applied Biomedical Research and Training Center, Evangelismos General Hospital, University of Athens Medical School, 2Department of Pathology, Evangelismos General Hospital, 31st Department of Pathology, University of Athens Medical School, Athens, Greece Abstract: Combined resistive breathing (CRB) is the hallmark of obstructive airway disease pathophysiology. We have previously shown that severe inspiratory resistive breathing (IRB) induces acute lung injury in healthy rats. The role of expiratory resistance is unknown. The possibility of a load-dependent type of resistive breathing-induced lung injury also remains elusive. Our aim was to investigate the differential effects of IRB, expiratory resistive breathing (ERB), and CRB on healthy rat lung and establish the lowest loads required to induce injury. Anesthetized tracheostomized rats breathed through a two-way valve. Varying resistances were connected to the inspiratory, expiratory, or both ports, so that the peak inspiratory pressure (IRB) was 20%–40% or peak expiratory (ERB) was 40%–70% of maximum. CRB was assessed in inspiratory/expiratory pressures of 30%/50%, 40%/50%, and 40%/60% of maximum. Quietly breathing animals served as controls. At 6hours, respiratory system mechanics were measured, and bronchoalveolar lavage was performed for measurement of cell and protein concentration. Lung tissue interleukin-6 and interleukin-1β levels were estimated, and a lung injury histological score was determined. ERB produced significant, load-independent neutrophilia, without mechanical or permeability derangements. IRB 30% was the lowest inspiratory load that provoked lung injury. CRB increased tissue elasticity, bronchoalveolar lavage total cell, macrophage and neutrophil counts, protein and cytokine levels, and lung injury score in a dose-dependent manner. In conclusion, CRB load dependently deranges mechanics, increases permeability, and induces inflammation in healthy rats. ERB is a putative inflammatory stimulus for the lung. Keywords: resistive breathing, lung injury, inflammation

Cigarette smoking is one of the risk factors for coronary artery disease. Although conditioning decreases infarct size in hearts from healthy animals, comorbidities may render it ineffective. We investigated the effects of cigarette smoke (CS) exposure on intracellular myocardial signaling, infarct size after ischemia-reperfusion, and the potential interference with ischemic conditioning. Exposure of mice to CS increased blood pressure, caused cardiac hypertrophy, and upregulated the nitric oxide synthatse (NOS)/soluble guanylate cyclase (sGC)/cGMP pathway. To test the effect of CS exposure on the endogenous cardioprotective mechanisms, mice were subjected to regional myocardial ischemia and reperfusion with no further intervention or application of preconditioning (PreC) or postconditioning (PostC). Exposure to CS did not increase the infarction compared with the room air (RA)-exposed group. PreC was beneficial for both CS and RA vs. nonconditioned animals. PostC was effective only in RA animals, while the infarct size-limiting effect was not preserved in the CS group. Differences in oxidative stress markers, Akt, and endothelial NOS phosphorylation and cGMP levels were observed between RA and CS groups subjected to PostC. In conclusion, exposure to CS does not per se increase infarct size. The beneficial effect of ischemic PreC is preserved in mice exposed to CS, as it does not affect the cardioprotective signaling; in contrast, PostC fails to protect CS-exposed mice due to impaired activation of the Akt/eNOS/cGMP axis that occurs in parallel to enhanced oxidative stress.

Combined inspiratory and expiratory resistive breathing (CRB) is the landmark of obstructive airway disease. Inspiratory loading and CRB via tracheal banding are known to induce acute lung injury in previously healthy animals. The role of expiratory resistance remains unknown.Aim: To elucidate the differential effect of inspiratory, expiratory and combined resistive loading on healthy rat lung mechanics and inflammatory status and establish the lowest loads required to induce injury.Anesthetized tracheostomized wistar rats breathed through a two-way valve. A resistance was connected to the inspiratory, expiratory or both ports, so that measured peak inspiratory pressure was modulated to (IRB) 20-40% of maximum or peak expiratory to (ERB) 30-70%. Combined resistive breathing was studied in inspiratory/expiratory pressures of 30%/50%, 40%/50% and 40%/60% of maximum. Quietly breathing animals served as controls. At six hours, respiratory system mechanics were measured and bronchoalveolar lavage (BAL) was performed for cell and protein concentration counting. Both IRB and CRB increased tissue elasticity, BAL total cell, macrophage and neutrophil counts and protein content in a dose dependent manner. IRB 30% and CRB 40%/50% were identified as the lowest loads able to induce significant mechanical distortion and lung inflammation. ERB failed to induce significant changes, with the exception of BAL fluid neutrophilia. IRB and CRB, above 30% and 40%/50% respectively, dose-dependently derange mechanics, increase permeability and induce inflammation in healthy rats. ERB is a putative per se inflammatory stimulus for the lung.

Electronic cigarettes (e-cigarettes) are advertised as a new smoking cessation tool and an alternative to cigarette smoking, aiming to limit smoking-related illnesses. However, concerns about their safety and toxicity have been raised. In spite of widespread use, only a limited number of studies on their effects in the respiratory system exist. We aimed to compare the effects of e-cigarette vapor vs cigarette smoke in the lungs of mice. Methods: C57BL/6 mice were exposed for 3 days or 4 weeks to air, cigarette smoke (CS) or e-cigarette vapor from i) propylene glycol/vegetable glycerol (1:1; PGVG), ii) PGVG with flavor (tobacco blend 1%; PGVG-F), or iii) PGVG with nicotine (18 mg/ml; PGVG-N). Results: After 3 days, BALF cellularity was increased (mostly macrophages) in all groups compared to air-breathing mice. After 4 weeks, BALF cellularity was elevated only in the CS and PGVG-F mice. Using a forced oscillation technique we observed that 3 day exposure to PGVG resulted in increased tissue elasticity and airway resistance compared to air-exposed mice. In the 4 week groups, airway elasticity was increased only in the CS and PGVG-F. Airway resistance in response to metacholine was increased in the CS and PGVG-F groups. Markers of oxidative stress and inflammation (protein carbonyls, malondialdehyde, interleukin-1β and -6) after 4 weeks were increased only in CS mice. Conclusions: Our findings suggest that after prolonged use, e-cigarettes trigger milder inflammatory responses and respiratory perturbations and that the flavor added was responsible for most of the observed damage.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure–volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC–cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Background & Objectives: During exacerbations of chronic obstructive pulmonary disease (COPD), increased airway inflammation and the accompanying bronchoconstriction lead to significant airway narrowing, i.e. resistive breathing (RB).To isolate the effects of mechanical stressor (i.e. RB) from the accompanying lung inflammation we developed an experimental model of RBthat exhibits both increased inspiratory and expiratory airway resistance, via tracheal banding . Since hydrogen sulphide (H 2 S) plays an important role in vasodilation and inflammation we hypothesized that the administration of a H 2 S fast releasing donor, Na 2 S, will attenuate the lung inflammation. Methods: C57BL/6 mice were studied: 1. Sham operated (controls) 2. Tracheal banded (TB) mice (tracheal surface area restricted to 50% of initial) 3. Sham operated mice treated with injected Na 2 S (10mg/kg) 4. Mice treated with injected Na 2 S 20 min prior to tracheal banding. Results: The proteinexpression of the three H 2 S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3MST), was the same between the TB and sham operated group in the lung. However, the lung mRNA level of CBS was decreased by 57% in TB mice (p=0.009). Finally interleukin IL-6 and IL-1β, were attenuated by 50% (p=0.004)and 45% (p=0.02)in the group of TB mice treated with Na 2 S compared to the TB mice, whereas the expression of TNF-α was the same among the groups. Conclusions: H 2 S ameliorates lung inflammation caused by airway narrowing. Our model of TB might offer new insights by which resistive breathing can enhance inflammation in obstructive lung disease.