Reduced activity of histone deacetylase 2 (HDAC2) has been described in patients with chronic obstructive pulmonary disease (COPD), but the mechanisms resulting in decreased expression of this important epigenetic modifier remain unknown. Here, we employed several in vitro experiments to address the role of microRNAs (miRNAs) on the regulation of HDAC2 in endothelial cells. Manipulation of miRNA levels in human pulmonary artery endothelial cells (HPAEC) was achieved by using electroporation with anti-miRNAs and miRNA mimics. Target prediction software identified miR-223 as a potential repressor of HDAC2. In subsequent stimulation experiments using inflammatory cytokines known to be increased in patients with COPD, miR-223 was found to be significantly induced. Functional analysis demonstrated that overexpression of miR-223 decreased HDAC2 expression and activity in HPAEC. Conversely, HDAC2 expression and activity was preserved in anti-miR-223-treated cells. Direct miRNA-target interaction was confirmed by reporter gene assay. In a next step, reduced expression of HDAC2 was found to increase the levels of the chemokine fractalkine (CX3CL1). In vivo studies confirmed elevated expression levels of miR-223 in mice exposed to cigarette smoke and in emphysematous lung tissue from LPS-treated mice. Moreover, a significant inverse correlation of miR-223 and HDAC2 expression was found in two independent cohorts of COPD patients. These data emphasize that miR-223, the most prevalent miRNA in COPD, controls expression and activity of HDAC2 in pulmonary cells, which, in turn, might alter the expression profile of chemokines. This pathway provides a novel pathogenic link between dysregulated miRNA expression and epigenetic activity in COPD.

Introduction: Pulmonary hypertension is a common complication in patients with severe chronic obstructive pulmonary disease (COPD) but the pathogenesis of its development is not completely understood. MicroRNAs (miRNAs) are small, non-coding gene regulators that have been associated with the pathogenesis of pulmonary hypertension. Here we investigated the signature from miRNAs in lung tissue slides from patients with end-stage COPD with and without elevated pulmonary pressure. Methods: Paraffin-embedded lung tissue specimens from COPD patients undergoing lung volume reduction surgery (LVRS) or lung transplantation were used. Pulmonary hemodynamics was invasively assessed in all patients before surgery. miRNA isolation was performed using the recoverall total nucleic acid isolation kit (Ambion ®) and quantified by qPCR. Results: 11 miRNAs previously associated with the pathogenesis of pulmonary hypertension were analysed. Significantly decreased expression levels of miR-125a (-0.54±0.33 vs 0.01±0.65, p<0.01), miR-130a (-0.18±0.43 vs 0.54±0.66, p<0.01), miR-301a (4.49±0.63 vs 5.02±0.71, p<0.05) and miR-424-5p (10.37±0.7 vs 11.43±0.86, p<0.01) were detected in COPD patients with pulmonary hypertension as compared to normotensive controls. A significant inverse association was found between miR-125a, miR-130a and miR-424-5p and mean pulmonary arterial pressure. Conclusion: This is the first report on the miRNA profile of COPD patients with and without pulmonary hypertension showing that most miRNAs are downregulated in patients with PH-COPD. Further studies are needed to answer the question whether this miRNA profile can be employed as predictor for the presence of an elevated pulmonary pressure in COPD patients.

Background: Histone Deacetylase 2 (HDAC2) is a key epigenetic modifier whose expression and activity have been found downregulated in patients with chronic obstructive pulmonary disease (COPD). MicroRNAs (miRNAs) are a group of small, non-coding RNA molecules that have emerged as gene silencers. We hypothesized that miRNAs are involved in the regulation of HDAC2 activity and expression. Methods: Levels of miRNA and mRNA were quantified by quantitative PCR. Protein expression and activity of HDAC2 were measured by Western Blotting and HDAC-Glo assay. Manipulation of miRNA levels in human pulmonary endothelial cells (HPAEC) was achieved by using electroporation with either miRNA inhibitors or miRNA mimics. Interaction between miRNA-223 and HDAC2 promoter was assessed with a reporter gene assay. Results: Prediction software (miRWalk ®) identified miRNA-223 as a potential repressor of HDAC2 (miRNA-223 was recently found to be upregulated in COPD patients (Ezzie, M.E. et al., Thorax 2012; 67(2):122-131)). Stimulation experiments with inflammatory cytokines involved in the pathogenesis of COPD (e.g. IL-1β, IL-6 and TNF-α) induced the expression of miRNA-223 in HPAEC. Subsequent functional experiments demonstrated that overexpression of miRNA-223 decreased both HDAC2 expression levels and its activity in HPAEC. Direct miRNA-target interaction was confirmed by reporter gene assay. Conclusion: Our data suggest that miRNA-223, the most prevalent miRNA in COPD, controls expression and activity of HDAC2 in pulmonary cells. This pathway provides a novel pathogenetic link between dysregulated miRNA expression and epigenetic activity in COPD.

Levels of microRNAs (miRNAs) are increasingly assessed in biological fluids, for example, in samples obtained by bronchoalveolar lavage (BAL). "Post-collection kinetics" of miRNA expression levels, however, have not been investigated to date. In these experiments, we analyzed the dynamic expression profile of 5 different miRNAs (miR-17, miR-19b, miR-20b, miR-125a, and miR-223-3p) in BAL within the first 24 h following collection by routine bronchoscopy. miRNAs were quantified 0, 1, 4, 8, and 24 h after collection in samples that were kept at 4 °C or at room temperature. The expression of all five miRNAs was found to remain stable between the first 8 h after collection. 24 h after collection miRNAs faced substantial alterations in their expression profile. These data emphasize that BAL samples intended for further miRNA analysis can be handled at room temperature within the first 8 h after bronchoscopy.

Introduction MicroRNAs (miRs) are involved in the fine tuning of gene expression in cellular processes and can be quantified in plasma. miRs represent a distinct class of easily accessible biomarkers and may play a role in clarifying pathogenic pathways in diseases involving the vascular system, such as obstructive sleep apnea (OSA). Methods miRs were isolated from stored plasma samples of OSA patients who participated in a randomized controlled trial defining the effects of a two-week continuous positive airway pressure (CPAP) therapy withdrawal. The plasma concentrations of 14 miRs selected in a pilot experiment, were measured by qPCR. Results The mean oxygen desaturation index (ODI) change from baseline was 43.0/h (SD 14.5, n=14) in the CPAP withdrawal group and 0.2/h (SD 1.8, n=14) in the CPAP group. Compared to continuing CPAP therapy CPAP withdrawal was associated with a down-regulated plasma concentration of hsa-miR-502-3p by a factor of 2.0 (n=14, p=0.045). In contrast, the plasma concentration of hsa-miR-210, a key regulator in the response to hypoxia, did not change (n=14, p=0.137). Conclusion Short-term CPAP therapy withdrawal in OSA patients resulted in a statistically significant change in the plasma concentration of hsa-miR-502-3p. In vitro studies are necessary to identify possible targets of hsa-miR-502-3p and to substantiate the relevance of our finding in OSA.

Background:Severe mucosal tissue damage requiring efficient wound healing is a main feature of inflammatory bowel disease but excessive tissue repair promotes fibrosis. The clinical investigation of fibrosis is confined to the limited amount of biological material available from patients. This makes the establishment of a new animal model, a highly desirable goal. We investigated whether intestinal fibrosis occurs after heterotopic transplantation of small bowel resections in rats.Methods: Donor (Brown Norway or Lewis rats) small bowel resections were transplanted subcutaneously into the neck of recipients (Lewis rats). Grafts were explanted 2, 7, 14, and 21 days after transplantation.Results: Heterotopic intestinal transplants remained viable for 21 days. Rapid loss of crypt structures at day 2 after intestinal transplantation was followed by lymphocyte infiltration and obliteration of the intestinal lumen by fibrous tissue at day 21. Loss of the intestinal epithelium was confirmed by the lack of cytokeratin staining in immunohistochemistry. Collagen expression was increased with time after transplantation as confirmed by real-time PCRs, Elastica van Gieson, and Sirius Red staining. Lumen obliteration was connected with increased expression of potent mediators of fibrosis such as 56 integrin, interleukin (IL)-13, and transforming growth factor . Myofibroblast phenotype was demonstrated by the presence of both -smooth muscle actin and vimentin in the obliterated lumen.Conclusions: We established a method for heterotopic transplantation of small bowel resections. A variety of histologic and molecular features of fibrosis were observed in the heterotopic intestinal grafts which suggests, that this new in vivo model will be instrumental in studying pathogenesis and treatment of intestinal fibrosis.

Background: Respiratory therapy in cystic fibrosis (CF) consists of airway clearance, infection control, and reduction of airway inflammation. It is well recognized that physical activity as well as daily chest physiotherapy, enhance airway clearance. We investigated the effects of pulmonary rehabilitation, including physical activity and chest physiotherapy, on airway inflammation in children with CF. Methods: Eighteen children with stable CF (six females), aged 8.2-16.2 years, participating in a 3-week multidisciplinary inpatient rehabilitation program were recruited. Assessment at the beginning and the end of the program included clinical score, pulmonary function test, exhaled breath condensate (EBC) and sputum analysis. Sputum supernatant and EBC were analyzed for interleukin (IL)-1b, 6, 8, 10, 12, tumor necrosis factor-alpha (TNF-) and LTB4. Results: Median (IQR) symptom scores decreased from 19 [23] to 16 [21], P=0.005. Vital capacity and FVC increased significantly (P<0.05). However no difference was found for the total sputum cells and sputum as well as EBC cytokines between the two visits. Significant correlations were found for sputum IL-1 (+), IL-6 (-), and IL-8 (+) to total sputum cell count and neutrophils and for IL-8 to TNF-alpha. Conclusions: We have shown that a short-term inpatient rehabilitation for children with stable CF with intensive physical activity mainly improve subjective clinical symptoms and measures of lung function such as VC and FVC but does not influence airflow obstruction and airway inflammation as assessed by sputum and EBC analysis. Pediatr Pulmonol. 2010; 45:541-551. (C) 2010 Wiley-Liss, inc.

Reliable, noninvasive, and highly reproducible surrogate markers for the early detection of bronchiolitis obliterans syndrome (BOS) after lung transplantation are needed. It has been shown that exhaled nitric oxide (FeNO) is increased in transplant recipients with infection, lymphocytic bronchiolitis, and early BOS (1, 2). However, studies to date are heterogeneous with regards to their FeNO measurements as a variety of different single expiratory flow rates have been used (45–250 mL/s). Guidelines recommend standardized measurements of exhaled lower respiratory NO using a single expiratory flow rate of 50 mL/s (3). Nevertheless, the question remains unanswered how different flow rates influence FeNO measurements in lung transplant recipients, in particular, for the detection of BOS as airflow obstruction is due to obliteration of small peripheral airways. We studied pulmonary NO exchange dynamics in stable lung transplant recipients with or without BOS and healthy controls, comparing different exhalation flow rates (50–200 mL/s) and evaluating whether the use of different flow rates influence FeNO measurements. MATERIALS AND METHODS Adult double-lung transplant recipients were recruited. Patients were only included if clinically stable without evidence of acute rejection or acute infection and a stable lung function over the 3 months preceding study inclusion. Recipients were divided into two groups: stable recipients with forced expiratory volume in 1 sec (FEV1) more than 90% of baseline value (group A) and recipients with BOS more than or equal to 1 (group B). Controls had no medical history of lung disease or atopy, any respiratory infection, and normal lung function tests. Smokers were excluded from the study. FeNO measurements were performed according to the published guidelines with a CLD 88-NO-analyser of Eco Medics (Dürnten, Switzerland), using the chemiluminescent NO measuring method (3). Each subject inhaled NO-free air to the total lung capacity and expired as long as possible at four different flow rates (50, 100, 150, and 200 mL/s) against a resistance of 10 cm H2O/L/s as previously described (4). Four different flow or pressure restrictors are designed to guarantee a laminar flow of desired flow rate with constant resistance of 10 cm H2O/L/s for velum closure. The flow was measured with an ultrasound flow meter, and biofeedback on a computer screen was used to maintain the desired flow. The archived flow with the corresponding NO and exhaled volume was registered. The dead space of the flow meter was 20 mL. The end-expiratory FeNO in the plateau phase (75%–95% of the exhalation cycle) was used for calculations. Three measurements were taken at each flow rate. Spirometry was performed on the same day before the NO measurements according to standard laboratory protocols (5, 6). The Mann-Whitney test was used for the statistical calculation. The value of P less than 0.05 was considered statistically significant. The local Research Ethics Committee approved the study. Written informed consent was obtained from all subjects. RESULTS Thirty transplant recipients and 16 controls were included. Twenty-two recipients had a FEV1 more than 90% of baseline value without the evidence of BOS (group A) and eight recipients were in BOS stage more than or equal to 1, FEV1 1.7 L (range, 1.1–2.3; group B). BOS stages of recipients in group B were as follows: BOS stage 1, n=4; BOS stage 2, n=1; BOS stage 3, n=3. Age, gender distribution, and time after transplantation were not significantly different between groups. As shown in Figure 1, FeNO measurements were similar in groups A and B and the controls. Moreover, a strong and significant correlation of FeNO measured with different flows was detected, with a high correlation coefficient in the two groups of transplant recipients. Although the correlation was lower in the healthy controls, it is still significant.FIGURE 1.: End-expiratory FeNO at different fixed exhalation flow rates. The average end-expiratory FeNO from three consecutive measurements at each of four fixed exhalation flow rates (VE=50, 100, 150, and 200 mL/s) is shown for each control (black dots), lung transplant recipients without BOS (group A, white dots), and lung transplant recipients with BOS more than 1 (group B; BOS 1, black triangles; BOS 2, white triangles; BOS 3, inverted triangles). The mean FeNO of each group is depicted as a line. Results as calculated using the Mann-Whitney test are given above the groups compared. BOS, Bronchiolitis Obliterans Syndrome; FeNO, exhaled nitric oxide; ns, not significant.DISCUSSION Our study demonstrates that pulmonary NO exchange dynamics in stable lung transplant recipients with and without BOS and healthy controls are similar even if different exhalation flow rates for FeNO measurements are used. FeNO measurements have been proposed as a useful noninvasive tool to assess lung graft function as a part of the posttransplantation management. To date, FeNO measurements in lung transplant recipients were conducted with the use of constant flow rates. Yet, the use of constant flow rates does not allow to differentiate whether the endogenous production of NO originates in the proximal or peripheral airways of the lungs (4). In conclusion, the goal of our study was a proof of principle, that is, the similarity of FeNO measurements in stable lung transplant recipients with and without BOS and healthy subjects irrespectively of the flow rates used. Further studies are warranted to evaluate the use of different flow rates in lung transplant recipients particularly with early stages of BOS, because FeNO measurements at different flow rates should allow to differentiate between alveolar and bronchial NO production. Markus Hofer Christian Benden Thomas Rechsteiner Annette Boehler Division of Pulmonary Medicine University Hospital Zurich Zurich, Switzerland

Extracorporeal photopheresis has anti-inflammatory properties. The development of pulmonary fibrosis includes inflammatory episodes. This study evaluates effects of extracorporeal photopheresis in experimental pulmonary fibrosis. The bleomycin model of pulmonary fibrosis was used. Two groups of 4 rats received intratracheal bleomycin to induce fibrosis. The treatment group received infusions of photochemically treated leukocytes harvested from syngeneic animals. All animals were sacrificed at day 21 after fibrosis induction and analyzed with respect to lung histology and hydroxyproline content, cellular composition of bronchoalveolar lavages, serum and lavage concentrations of transforming growth factor-beta, interferon-gamma, and interleukin-10, and expression of selected genes in the lung. Interleukin-10 and transforming growth factor-beta protein concentrations increased in the plasma of treated animals, whereas the interferon-gamma protein concentration was higher in bronchoalveolar lavages. Interferon-gamma gene expression was up-regulated in the lung tissue of treated animals. No significant differences between treated and untreated animals were found with respect to hydroxyproline, histology, and lavage cell count. To conclude, extracorporeal photopheresis has positive molecular effects but does not attenuate experimental lung fibrosis with respect to histology, hydroxyproline, and lavage cell count in the applied treatment regimen. Further investigations of extracorporeal photopheresis in experimental pulmonary fibrosis are justified.

In cystic fibrosis (CF) lung disease, exhaled nitric oxide (FeNO) is not raised, but rather is normal or even decreased when measured at a single expiratory flow. FeNO measurements at several flow rates allow differentiation between alveolar and bronchial nitric oxide (NO) production. Extended FeNO measurements therefore should be useful to localize the FeNO deficit in CF airways. FeNO was measured in stable CF adults with moderate lung disease and in healthy controls. Bronchial NO fluxes (J NO,Br ) and alveolar NO concentrations (C Alv ) were calculated from FeNO measurements at flow rates of 100, 150 and 200 ml/s using a method previously described. Thirty-two adults were included in the study, 12 of whom had CF. CF adults had significantly lower FeNO values at all flow rates. The median J NO,Br was significantly lower in CF adults than in healthy controls [0.31 nl/s (range = 0.11–0.63) vs. 0.70 nl/s (0.27–3.52); P < 0.001], while the median C Alv was similar in both groups [1.7 ppb (0.3–3.9) vs. 1.2 (0.1–5.2)]. Pulmonary NO exchange did not differ significantly between subgroups of CF patients with and without chronic Pseudomonas aeruginosa infection. No significant correlation was detectable between FEV 1 /VC and J NO,Br and C Alv , respectively. Extended FeNO measurements can separate alveolar and bronchial NO outputs in CF adults. The lower FeNO in adults with moderate to severe CF lung disease is likely to be the result of lower bronchial NO output.

Oxidative stress occurs at altitude, and physical exertion might enhance this stress. In the present study, we investigated the combined effects of exercise and moderate altitude on redox balance in ten endurance exercising biathletes, and five sedentary volunteers during a 6-week-stay at 2,800 m. As a marker for oxidative stress, hydrogen peroxide (H2O2) was analyzed by the biosensor measuring system Ecocheck™, and 8-iso prostaglandin F2α (8-iso PGF2α) was determined by enzyme immunoassay in exhaled breath condensate (EBC). To determine the whole blood antioxidative capacity, we measured reduced glutathione (GSH) enzymatically using Ellman's reagent. Exercising athletes and sedentary volunteers showed increased levels of oxidative markers at moderate altitude, contrary to our expectations; there was no difference between both groups. Therefore, all subjects' data were pooled to examine the oxidative stress response exclusively due to altitude exposure. H2O2 levels increased at altitude and remained elevated for 3 days after returning to sea level (p ≤ 0.05). On the other hand, 8-iso PGF2α levels showed a tendency to increase at altitude, but declined immediately after returning to sea level (p ≤ 0.001). Hypoxic exposure during the first day at altitude resulted in elevated GSH levels (p ≤ 0.05), that decreased during prolonged sojourn at altitude (p ≤ 0.001). In conclusion, a stay at moderate altitude for up to 6 weeks increases markers of oxidative stress in EBC independent of additional endurance training. Notably, this oxidative stress is still detectable 3 days upon return to sea level.

Background. Ciprofloxacin is widely used to treat respiratory tract infections. Like other fluoroquinolones, ciprofloxacin has immunomodulatory effects; however, it is unknown whether these effects are beneficial in the setting of lung transplantation. We investigated potential immunomodulatory effects of ciprofloxacin in a model of posttransplant bronchiolitis obliterans. Methods. The heterotopic tracheal transplantation model in rats was used. Three groups received ciprofloxacin and underwent different immunosuppressive regimens of cyclosporine A, that is, no immunosuppression, insufficient immunosuppression, or low-dose immunosuppression. Three groups underwent the same immunosuppressive regimen but had no ciprofloxacin treatment. Tracheas were harvested after 21 days and examined with respect to histology and expression of selected cytokines. Results. The allografts of animals treated with ciprofloxacin showed less airway obliteration compared with allografts of untreated animals. When combimed with low-dose immunosuppression ciprofloxacin showed beneficial effects in preventing airway obliteration and rejection of the respiratory epithelium. Cytokine gene expression of the allografts treated with ciprofloxacin was higher with respect to transforming growth factor-&bgr; and equal with respect to tumor necrosis factor-&agr; and interferon-&ggr; compared with controls. When applied in combination with cyclosporine A, ciprofloxacin lowered the expression of transforming growth factor-&bgr; and tumor necrosis factor-&agr; and increased interferon-&ggr; expression. Conclusion. Ciprofloxacin attenuates airway rejection after tracheal transplantation. Genetic expression of mediators that are known to play an important role in mediating rejection in this model supports an immunomodulatory and antifibrotic role of ciprofloxacin. These findings suggest that further clinical studies are needed to investigate whether ciprofloxacin in addition to its bactericidal effect might be beneficial in the treatment of human posttransplant bronchiolitis obliterans.