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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.

S100B protein, an acknowledged biomarker of brain injury, has been reported to be increased in hemorrhagic shock. Also, acute hemorrhage is associated with inflammatory response. The aim of this study was to investigate the concentrations of serum S100B and the potential relationships with interleukin 6 (IL-6), severity of tissue hypoperfusion, and prognosis in patients admitted for surgical control of severe hemorrhage. Patients undergoing elective abdominal aortic aneurysm surgery participated as control subjects. Serum samples were drawn before, at the end of surgery, and after 6 and 24 h. Sixty-four patients with severe hemorrhage (23 trauma and 41 nontrauma) and 17 control subjects were included. Increased preoperative concentrations of S100B protein (1.70 ± 2.13 and 0.81 ± 1.23 μg/L) and IL-6 (241 ± 291 and 226 ± 238 pg/mL) were found in patients with traumatic and nontraumatic reason, respectively, and remained elevated throughout 24 h. Compared with nontrauma, trauma patients exhibited higher preoperative S100B levels (P < 0.05). Overall mortality was 47%. In control subjects, preoperative S100B and IL-6 levels were within normal limits and increased at the end of surgery (P < 0.001 and P < 0.01, respectively). Preoperative S100B correlated with IL-6 (r = 0.78, P < 0.01), arterial lactate (r = 0.50, P < 0.01), pH (r = -0.45, P < 0.01), and bicarbonate (r = -0.40, P < 0.01). Multiple analysis revealed that preoperative S100B in trauma and lactate in nontrauma patients were independently associated with outcome. In predicting death, preoperative S100B yielded receiver operator characteristics curve areas of 0.75 for all patients and 0.86 for those with trauma. These results indicate that severe hemorrhage in patients without brain injury is associated with increased serum levels of S100B, which correlates with IL-6 and tissue hypoperfusion. Moreover, the predictive ability of S100B for mortality, suggests that it could be a marker of potential clinical value in identifying, among patients with severe hemorrhage, those at greater risk for adverse outcome.

Purpose Endothelial protein C receptor (EPCR) is expressed mainly in endothelial cells and is involved in regulation of the cytoprotective and anticoagulant pathways of protein C. We assessed whether haplotypes in the EPCR gene modify the risk of severe sepsis and/or septic shock (SS/SS) development in critically ill patients. Methods Three polymorphisms in the EPCR gene were genotyped in 389 Caucasian critically ill patients, hospitalized in the intensive care units of two major hospitals in Athens, Greece. Multivariate logistic regression analysis controlling for age, acute physiology and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores, sex, and diagnosis was performed to determine the effect of haplotypes H1 and H3 in the EPCR gene on the development of SS/SS. Results H2 carriers versus all other genotypes combined had a nonsignificant excess of SS/SS (p = 0.087). SS/SS occurred in 38.8 % of critically ill patients carrying minor alleles belonging to both H1 and H3 haplotypes, in 58.0 % of H1 carriers, 64.3 % of H3 carriers, and 65.2 % of patients carrying all common alleles (H2). Compared with H2 carriers, the odds ratios (OR) for developing SS/SS were 0.34 [95 % confidence interval (CI) 0.16–0.76, p = 0.008] for simultaneous H1 and H3 carriers, 0.65 (95 % CI 0.37–1.13, p = 0.123) for H1 carriers, and 0.82 (95 % CI 0.39–1.70, p = 0.590) for H3 carriers. Conclusions Our results indicate that simultaneous carriers of minor alleles belonging to both the H1 and H3 haplotypes may be at reduced risk of developing SS/SS in this cohort of critically ill patients.

Pulmonary endothelium is a major metabolic organ affecting pulmonary and systemic vascular homeostasis. Brain death (BD)-induced physiologic and metabolic derangements in donors' lungs, in the absence of overt lung pathology, may cause pulmonary dysfunction and compromise post-transplant graft function. To explore the impact of BD on pulmonary endothelium, we estimated pulmonary capillary endothelium-bound (PCEB)-angiotensin converting enzyme (ACE) activity, a direct and quantifiable index of pulmonary endothelial function, in eight brain-dead patients and ten brain-injured mechanically ventilated controls. No subject suffered from acute lung injury or any other overt lung pathology. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate (3)H-benzoyl-Phe-Ala-Pro, under first order reaction conditions, and calculated lung functional capillary surface area (FCSA). Substrate %M (35 ± 6.8%) and v (0.49 ± 0.13) in BD patients were decreased as compared to controls (55.9 ± 4.9, P = 0.033 and 0.9 ± 0.15, P = 0.033, respectively), denoting decreased pulmonary endothelial enzyme activity at the capillary level; FCSA, a reflection of endothelial enzyme activity per vascular bed, was also decreased (BD patients: 1,563 ± 562 mL/min vs 4,235 ± 559 in controls; P = 0.003). We conclude that BD is associated with subtle pulmonary endothelial injury, expressed by decreased PCEB-ACE activity. The applied indicator-dilution type technique provides direct and quantifiable indices of pulmonary endothelial function at the bedside that may reveal the existence of preclinical lung pathology in potential lung donors.

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.

Resistive breathing is associated with large negative intrathoracic pressures. Increased mechanical stress induces high-permeability pulmonary edema and lung inflammation.To determine the effects of resistive breathing on the healthy lung.Anesthetized rats breathed through a two-way nonrebreathing valve. The inspiratory line was connected to a resistance setting peak inspiratory tracheal pressure at 50% of maximum (inspiratory resistive breathing), while 100% oxygen was supplied to prevent hypoxemia. Quietly breathing animals (100% oxygen) served as controls. Lung injury was evaluated after 3 and 6 hours of resistive breathing.After both 3 and 6 hours of resistive breathing, lung permeability was increased, as assessed by (99m)Tc-diethylenetriaminepentaacetic acid scintigraphy and Evans blue dye extravasation. Tissue elasticity, measured on the basis of static pressure-volume curves and by the low-frequency forced oscillation technique, was also increased. After both 3 and 6 hours of resistive breathing, gravimetric measurements revealed the presence of pulmonary edema and analysis of bronchoalveolar lavage showed increased total protein content, whereas the total cell count was elevated only after 6 hours of resistive breathing. Cytokine levels were assessed in bronchoalveolar lavage fluid and lung tissue by ELISA and were increased after 6 hours compared with controls. Western blot analysis showed early activation of Src kinase via phosphorylation (at 30 min), and Erk1/2 and IκBα (nuclear factor-κB inhibitor) were phosphorylated at 3 and 6 hours. Pathology revealed the presence of lung injury after resistive breathing.Resistive breathing induces acute lung injury and inflammation.

OBJECTIVE We hypothesized that pretreatment with atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, confers protection against lung injury caused by high-stretch mechanical ventilation. METHODS Twenty-four isolated sets of normal rabbit lungs were utilized. Treated animals received atorvastatin (20 mg/kg body weight/day by mouth) for 3 days before surgery. Lungs were perfused constantly (300 mL/min) and ventilated for 1 hr with pressure-control ventilation at either 23 (high pressure; resulting in tidal volume approximately 22 mL/kg) or 11 (low pressure; tidal volume approximately 10 mL/kg) cm H2O peak inspiratory pressure and positive end-expiratory pressure of 3 cm H2O. Four groups were examined: high pressure-no statin, high pressure-statin pretreatment, low pressure-no statin, and low pressure-statin pretreatment. RESULTS The high-pressure-no statin group sustained more damage than the low-pressure groups. In high-pressure groups, lungs of statin-pretreated vs. no statin-pretreated animals sustained a significantly lower increase in ultrafiltration coefficient (an accurate marker of alveolar capillary permeability; high-pressure-statin pretreatment vs. high-pressure-no statin, -0.013 +/- 0.017 g/min/mm Hg/100g vs. 1.723 +/- 0.495 g/min/mm Hg/100g; p < .001), lower weight gain (i.e., less edema formation; 4.62 +/- 1.50 grams vs. 17.75 +/- 4.71 grams; p = .005), improved hemodynamics (i.e., lower increase in mean pulmonary artery pressure; 0.56 +/- 0.51 mm Hg vs. 5.62 +/- 1.52 mm Hg; p = .04), lower protein concentration in bronchoalveolar lavage fluid (p < .001), and fewer histologic lesions (p = .013). Apoptosis of lung parenchyma cells was not different (p = .97). There was no difference between low-pressure-statin pretreatment and low-pressure-no statin groups regarding these outcomes. CONCLUSION In this model, atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, attenuates lung injury caused by high-stretch mechanical ventilation.

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC alpha1 and beta1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the beta1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in beta1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased alpha1 and beta1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-alpha was required for the LPS-triggered reduction in sGC protein levels, as no change in alpha1 and beta1 levels was observed in TNF-alpha knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

Angiopoietin (Ang)-2 is an endothelium-specific growth factor, regulated by proinflammatory stimuli, that destabilizes vascular endothelium and increases vascular leakage; consequently, Ang-2 may contribute to sepsis pathophysiology. We have studied 1) serum Ang-2 levels in critically-ill patients and investigated potential relationships with inflammatory mediators and indices of disease severity and 2) the effect of sepsis-related inflammatory mediators on Ang-2 production by lung endothelium in vitro.Prospective clinical study followed by cell culture studies.General intensive care unit and research laboratory of a university hospital.Human and bovine lung microvascular endothelial cells and 61 patients (32 men). Patients were grouped according to their septic stage as having: no systemic inflammatory response syndrome (n = 6), systemic inflammatory response syndrome (n = 8), sepsis (n = 16), severe sepsis (n = 18), and septic shock (n = 13).Cells were exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6.Patients' serum Ang-2 levels were significantly increased in severe sepsis as compared with patients with no systemic inflammatory response syndrome or sepsis (p.05 by analysis of variance). Positive linear relationships were observed with: serum tumor necrosis factor-alpha (rs = 0.654, p.001), serum interleukin-6 (rs = 0.464, p.001), Acute Physiology and Chronic Health Evaluation II score (rs = 0.387, p.001), and Sequential Organ Failure Assessment score (rs = 0.428, p.001). Multiple regression analysis revealed that serum Ang-2 is mostly related to serum tumor necrosis factor-alpha and severe sepsis. Treatment of human lung microvascular endothelial cells with all mediators resulted in a concentration-dependent Ang-2 reduction. Treatment of bovine lung microvascular endothelial cells with lipopolysaccharide and tumor necrosis factor-alpha increased Ang-2 release, and interleukin-6 reduced basal Ang-2 levels.First, patients' serum Ang-2 levels are increased during severe sepsis and associated with disease severity. The strong relationship of serum Ang-2 with serum tumor necrosis factor-alpha suggests that the latter may participate in the regulation of Ang-2 production in sepsis. Second, inflammatory mediators reduce Ang-2 release from human lung microvascular endothelial cells, implying that this vascular bed may not be the source of increased Ang-2 in human sepsis.

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.

The serine protease activated protein C (APC) possesses prominent anticoagulant and anti-inflammatory actions. In this study, we investigatedthe effect of inhaled recombinant human (rh) APC in a murine lung injury model. Animals inhaled 10 mg of Pseudomonas lipopolysaccharide(LPS) in 3 mL normal saline (NS); 30 min prior to LPS, mice were pretreated with inhaled rhAPC (4 mg/3 mL NS; APC+LPS group) or NS (LPSgroup). A control animal group inhaled vehicle (NS) twice. 24 h later, total cells and cell-types, protein content, and the cytokines tumor necrosisfactor-α, interleukin (IL)-6, macrophage inflammatory protein-1α, and mouse keratinocyte-derived chemokine (a homolog of human IL-8) wereestimated in bronchoalveolar lavage fluid (BALF). Lung pathology given as total histology score (THS), wet/dry lung weight ratios, and lungvascular cell adhesion molecule (VCAM)-1 expression were additionally assessed. rhAPC inhalation attenuated the aerosolized LPS-inducedincreases of: total cells, neutrophils and macrophages in BALF, lung tissue VCAM-1 protein levels, and THS. Total protein levels and cytokines inBALF, and wet/dry weight ratios were increased in the LPS group, but rhAPC pretreatment did not significantly alter the LPS-induced responses.In conclusion, in this murine septic model of lung injury, inhaled rhAPC appears to attenuate lung inflammation, without reversing the observedincreases in lung permeability and BALF cytokines. This effect may be associated with leukocyte trafficking modifications, related, at least in part,to VCAM-1 reduction.© 2006 Elsevier Inc. All rights reserved.

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 —Pulmonary capillary endothelium-bound (PCEB) angiotensin-converting ectoenzyme (ACE) activity alteration is an early, sensitive, and quantifiable lung injury index in animal models. We hypothesized that (1) PCEB-ACE alterations can be found in patients with acute lung injury (ALI) and (2) PCEB-ACE activity correlates with the severity of lung injury and may be used as a quantifiable marker of the underlying pulmonary capillary endothelial dysfunction. Methods and Results —Applying indicator-dilution techniques, we measured single-pass transpulmonary hydrolysis of the synthetic ACE substrate 3 H-benzoyl-Phe-Ala-Pro (BPAP) in 33 mechanically ventilated, critically ill patients with a lung injury score (LIS) ranging from 0 (no lung injury) to 3.7 (severe lung injury) and calculated the kinetic parameter A max /K m . Both parameters decreased early during the ALI continuum and were inversely related to APACHE II score and LIS. Hydrolysis decreased with increasing cardiac output (CO), whereas 2 different patterns were observed between CO and A max /K m . Conclusions —PCEB-ACE activity decreases early during ALI, correlates with the clinical severity of both the lung injury and the underlying disease, and may be used as a quantifiable marker of underlying pulmonary capillary endothelial dysfunction.

Chronic obstractive pulmonary disease (COPD) in men has been associated with testosterone deficiency, known as the late-onset hypogonadism. Prostate cancer becomes more prevalent when testosterone values decline in males. We sought to determine endocrinological derangements that may affect PSA values in male patients with COPD.A total of 69 male patients with COPD and 82 healthy volunteers were divided into subgroups according to: their age: (i) ≤60 years and (ii)60 years; or disease severity: (i) FEV150% and (ii) FEV1≥50% predicted.There was a significant reduction in total and free testosterone in patients with COPD. Patients with COPD aged60 years had significantly lower free PSA compared to the control group.Alterations of the male hormonal status in COPD are related with older age (60 years) and poorer lung function (FEV150% predicted). This may have implications for the use of the PSA-based screening tests in the elderly male population with COPD.

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.

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.

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White ( n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [3H]benzoyl-Phe-Ala-Pro (BPAP) and the 5′-nucleotidase (NCT) substrate [14C]AMP were estimated, and the first-order reaction parameter A max/ K m, where A max is the product of enzyme mass and the catalytic rate constant and K m is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A max/ K m were reduced in WHHL (1.69 ± 0.16 vs. 2.9 ± 0.44 and 599 ± 69 vs. 987 ± 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K m and A max values were estimated separately under mixed-order reaction conditions. No differences in K m values were found (9.79 ± 1 vs. 9.9 ± 1.31 μM), whereas WHHL rabbit A max was significantly decreased (5.29 ± 0.88 vs. 7.93 ± 0.8 μmol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.

The pulmonary vascular endothelium participates in various important physiologic processes, such as synthesis, release and degradation of hormones and vasoactive peptides, prostaglandin synthesis, release and uptake, lipid processing, xenobiotic metabolism, and antithrombogenic-thrombolytic activities (Orfanos and Catravas, 1993). Enzymes responsible for many of these functions are located on the luminal surface of the capillaries (ectoenzymes). Due to their location, they are directly accessible to blood-borne substrates and their activities may be measured in vivo by means of indicator-dilution type techniques. Among these enzymes, angiotensin-converting enzyme (ACE) a major regulator of systemic blood pressure has been extensively studied in animals: monitoring pulmonary capillary endothelium-bound (PCEB) ACE activity in vivo under normal conditions allows estimations of the dynamically perfused capillary surface area (DPCSA; Orfanos et al., 1994), whereas under toxic conditions provides an early index of lung injury. We have initiated similar studies in man, in an effort to validate the use of the aforementioned techniques as a tool of pulmonary endothelial function assessment in the normal and injured human lung.

We present images obtained immediately pre-mortem from a 60-year-old woman with long-standing pulmonary alveolar microlithiasis. Posterior– anterior digital chest radiograph (Figure 1A) exhibits the characteristic bilateral diffuse micronodular ‘‘sandstorm’’ radiographic pattern. Chest computed tomography (Figure 1B) shows bilateral confluent micronodular parenchymal and pleural calcification (left; soft-tissue window; white arrows), and interstitial thickening/fibrosis (right; lung parenchymal window; additional opacities). Whole-body 99mTechenetium-methylene diphosphonate scintigraphy (Figure 1C) shows diffusely increased radiotracer uptake over the lungs (arrows). Microlithiasis has been linked to mutations in the alveolar phosphate-sodium co-transporter SLC34A2 (1). The pathophysiology of calcium phosphate deposition in the alveolar spaces is reviewed elsewhere (2).