Ellen Drost

Oxidative stress and airway inflammation in severe exacerbations of COPD

Background: A study was undertaken to assess both oxidative stress and inflammation in the lungs of patients with chronic obstructive pulmonary disease (COPD) during severe and very severe exacerbations compared with those with stable COPD, healthy smokers, and non-smokers. Two sites within the lungs were compared: the large airways (in sputum) and the peripheral airways (by bronchoalveolar lavage (BAL)). Methods: BAL fluid cell numbers and levels of tumour necrosis factor (TNFα) and interleukin (IL)-8 were measured as markers of airway inflammation and glutathione (GSH) levels as a marker of antioxidant status. Nuclear translocation of the pro-inflammatory transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) were also measured by electromobility shift assay in BAL fluid leucocytes and lung biopsy samples. Results: Influx of inflammatory cells into the peripheral airways during exacerbations of COPD was confirmed. Increased IL-8 levels were detected in BAL fluid from patients with stable COPD compared with non-smokers and healthy smokers, with no further increase during exacerbations. In contrast, IL-8 levels in the large airways increased during exacerbations. GSH levels were increased in the BAL fluid of smokers (444%) and patients with stable COPD (235%) compared with non-smokers and were reduced during exacerbations (severe 89.2%; very severe 52.3% compared with stable COPD). NF-κB DNA binding in BAL leucocytes was decreased in healthy smokers compared with non-smokers (41.3%, n = 9, p<0.001) but did not differ in COPD patients, whereas AP-1 DNA binding was significantly decreased during exacerbations of COPD. Conclusion: There is evidence of increased oxidative stress in the airways of patients with COPD that is increased further in severe and very severe exacerbations of the disease. This is associated with increased neutrophil influx and IL-8 levels during exacerbations.

Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation.

Background There has been concern regarding risks from inhalation exposure to nanoparticles (NPs). The large number of particles requiring testing means that alternative approaches to animal testing are needed. Objectives We set out to determine whether short-term in vitro assays that assess intrinsic oxidative stress potential and membrane-damaging potency of a panel of metal oxide NPs can be used to predict their inflammogenic potency. Methods For a panel of metal oxide NPs, we investigated intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to lung epithelial cells, hemolysis, and inflammation potency in rat lungs. All exposures were carried out at equal surface area doses. Results Only nickel oxide (NiO) and alumina 2 caused significant lung inflammation when instilled into rat lungs at equal surface area, suggesting that these two had extra surface reactivity. We observed significant free radical generation with 4 of 13 metal oxides, only one of which was inflammogenic. Only 3 of 13 were significantly hemolytic, two of which were inflammogenic. Conclusions Potency in generating free radicals in vitro did not predict inflammation, whereas alumina 2 had no free radical activity but was inflammogenic. The hemolysis assay was correct in predicting the proinflammatory potential of 12 of 13 of the particles examined. Using a battery of simple in vitro tests, it is possible to predict the inflammogenicity of metal oxide NPs, although some false-positive results are likely. More research using a larger panel is needed to confirm the efficacy and generality of this approach for metal oxide NPs.

Vascular Dysfunction in Chronic Obstructive Pulmonary Disease

RATIONALE Cardiovascular disease is a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), which may in part be attributable to abnormalities of systemic vascular function. It is unclear whether such associations relate to the presence of COPD or prior smoking habit. OBJECTIVES To undertake a comprehensive assessment of vascular function in patients with COPD and healthy control subjects matched for smoking history. METHODS Eighteen men with COPD were compared with 17 healthy male control subjects matched for age and lifetime cigarette smoke exposure. Participants were free from clinically evident cardiovascular disease. MEASUREMENTS AND MAIN RESULTS Pulse wave velocity and pulse wave analysis were measured via applanation tonometry at carotid, radial, and femoral arteries. Blood flow was measured in both forearms using venous occlusion plethysmography during intrabrachial infusion of endothelium-dependent vasodilators (bradykinin, 100-1,000 pmol/min; acetylcholine, 5-20 microg/min) and endothelium-independent vasodilators (sodium nitroprusside, 2-8 microg/min; verapamil, 10-100 microg/min). Tissue plasminogen activator (t-PA) was measured in venous plasma before and during bradykinin infusions. Patients with COPD have greater arterial stiffness (pulse wave velocity, 11 +/- 2 vs. 9 +/- 2 m/s; P = 0.003; augmentation index, 27 +/- 10 vs. 21 +/- 6%; P = 0.028), but there were no differences in endothelium-dependent and -independent vasomotor function or bradykinin-induced endothelial t-PA release (P > 0.05 for all). CONCLUSIONS COPD is associated with increased arterial stiffness independent of cigarette smoke exposure. However, this abnormality is not explained by systemic endothelial dysfunction. Increased arterial stiffness may represent the mechanistic link between COPD and the increased risk for cardiovascular disease associated with this condition.

Nanoparticle-driven DNA damage mimics irradiation-related carcinogenesis pathways

The epidemiological association between cancer and exposure to ambient air pollution particles (particles with a 50% cut-off aerodynamic diameter of 10 μm (PM10)) has been related to the ability of PM10 and its constituent nanoparticles (NPs) to cause reactive oxidative species (ROS)-driven DNA damage. However, there are no data on the molecular response to these genotoxic effects. In order to assess whether PM10, NP and ROS-driven DNA damage induce carcinogenesis pathways, A549 cells were treated with tert-butyl-hyperperoxide (Tbh), urban dust (UD), carbon black (CB), nanoparticulate CB (NPCB), benzo(a)pyrene (BaP) and NPCB coated with BaP for ≤24 h. Single- and double-strand breakage of DNA was determined by comet assay; cell cycle status was analysed using flow cytometry. Nuclear extracts or acid-extracted histones were used for Western blot analysis of p-ser15-p53 (p53 phosphorylated at ser15), p53 binding protein (53BP) 1, phospho-histone H2A.X (p-H2A.X) and phospho-BRCA1 (p-BRCA1). UD caused both single- and double-strand DNA breaks, while other tested NPs caused only single-strand DNA breaks. NPs significantly altered cell cycle kinetics. Tbh enhanced p-H2A.X after 1 and 6 h (2.1- and 2.2-fold, respectively). NP increased 53BP1 expression at 1 h (2.4–8.7-fold) and p-BRCA1 at 1–6 h. N-acetylcysteine blocked NP-driven p-ser15-p53 response. In conclusion, nanoparticles and reactive oxidative species induce DNA damage, activating p53 and proteins related to DNA repair, mimicking irradiation-related carcinogenesis pathways.

Apocynin increases glutathione synthesis and activates AP‐1 in alveolar epithelial cells

Apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) is a potent intracellular inhibitor of superoxide anion production in neutrophils. In this study, we studied the effect of apocynin on the regulation of the antioxidant glutathione (GSH) and activation of the transcription factor AP‐1 in human alveolar epithelial cells (A549). Apocynin enhanced intracellular GSH by increasing γ‐glutamylcysteine synthetase activity in A549 cells. Apocynin also increased the expression of γ‐GCS heavy subunit mRNA. This was associated with increased AP‐1 DNA binding as measured by the electrophoretic mobility shift assay. These data indicate that apocynin displays antioxidant properties, in part, by increasing glutathione synthesis through activation of AP‐1.

Nacystelyn inhibits oxidant-mediated interleukin-8 expression and NF-κB nuclear binding in alveolar epithelial cells

Nacystelyn (NAL), a recently developed lysine salt of N-acetyl-L-cytokine (NAC) has mucolytic and antioxidant properties. In this study, we investigated the effect of NAL upon oxidant-mediated interleukin (IL)-8 release and the activation of the redox-sensitive transcription factors AP-1, NF-kappaB, and C/EBP in a human alveolar epithelial cell line (A549). NAL (5 mM) enhanced intracellular glutathione (GSH) after 4 h and abolished H(2)O(2)-induced IL-8 release from A549 cells. This was associated with inhibition of NF-kappaB and C/EBP DNA-binding, measured by the Electrophoretic Mobility Shift Assay (EMSA). NAL also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyl transferase (CAT) reporter system, transfected into A549 cells. Supernatants obtained from H(2)O(2)-treated A549 cells induced chemotaxis of polymorphonuclear neutrophils, which could be inhibited by co-incubation with NAL. These data indicate that NAL may be used to modulate pro-inflammatory process by inhibiting cytokine release in the lungs and thus has therapeutic potential in inflammatory lung diseases.

The effects of N-acetylcysteine and glutathione on smoke-induced changes in lung phagocytes and epithelial cells

We studied the mechanism of the delay in neutrophil traffic in pulmonary microvasculature previously observed during cigarette smoking, the effect of cigarette smoke on lung phagocytes and epithelial cell function, and augmentation of the glutathione (GSH) antioxidant system using the thiol drug N-acetylcysteine. Using a micropore membrane system to mimic the dimensions of the average pulmonary capillary, we showed that cigarette smoke reduces cell deformability, increasing the difficulty experienced by the larger neutrophils in negotiating the smaller capillary segments, so delaying their passage during smoking. This effect is both diminished and recoverable by the addition of plasma, and by GSH in concentrations found in plasma. Cigarette smoke induces oxidative changes in both the cell membrane and the cell cytoskeleton, and diminishes the ability of neutrophils to release reactive oxygen intermediates. The injurious effect of oxidants can be measured in vitro by the detachment of 51Cr-radiolabeled alveolar epithelial cells grown in monolayers, an effect also diminished by the addition of GSH. Such epithelial cell detachment in vitro may be reflected as the epithelial permeability that occurs at an early stage in asymptomatic smokers. N-Acetylcysteine given orally (600 mg/day) increases both plasma and bronchoalveolar lavage GSH in normal subjects, but a sustained increase in plasma GSH requires higher dosage regimens in patients with chronic obstructive pulmonary disease (600 mg three times daily). Thus, the potential exists to enhance the antioxidant status of both plasma and the airspaces of the lungs against oxidant-induced injury.

Potential pro‐inflammatory effects of soluble E‐selectin upon neutrophil function

Appropriate recruitment of neutrophils to sites of infection or tissue injury is a key event in the inflammatory response. A number of studies have shown the critical role of selectins in tethering and rolling of neutrophils on vascular endothelium, as well as a more complex regulatory role, since they have the potential to alter leukocyte recruitment by triggering β2 integrin‐mediated adhesion. In this study, we report that in contrast to patients “at risk” of developing acute respiratory disease syndrome (ARDS), elevated plasma levels of soluble E‐selectin are found in patients with established disease. Since neutrophil granulocytes are implicated in ARDS pathogenesis, we have investigated the possibility of a link between elevated soluble plasma E‐selectin levels and disease progression by examining the effects of soluble recombinant E‐selectin (E‐zz) upon neutrophil function. In this paper, we describe the novel finding that exposure of neutrophils to E‐zz potentiates a number of neutrophil functions which may act to drive inflammatory processes. Although neutrophil deformability, an important parameter determining retention within the lung microvasculature, was not affected by E‐zz, neutrophil polarization was observed. In addition, neutrophil β2 integrin‐mediated adhesion was found to be augmented by E‐zz without alteration in levels of surface expression of αMβ2 or the “activation” reporter epitope defined by monoclonal antibody 24. Concomitantly with increased β2 integrin‐mediated adhesion, we observed an inhibition of formyl‐Met‐Leu‐Phe‐directed chemotaxis. Together with an augmentation of neutrophil reactive oxidant species production and release of superoxide anions, these data raise the possibility that soluble E‐selectin exerts pro‐inflammatory effects upon neutrophil function at sites of inflammation, thereby exacerbating disease processes.

Genome-wide mRNA expression profiling in vastus lateralis of COPD patients with low and normal fat free mass index and healthy controls

BackgroundChronic Obstructive Pulmonary Disease (COPD) has significant systemic effects beyond the lungs amongst which muscle wasting is a prominent contributor to exercise limitation and an independent predictor of morbidity and mortality. The molecular mechanisms leading to skeletal muscle dysfunction/wasting are not fully understood and are likely to be multi-factorial. The need to develop therapeutic strategies aimed at improving skeletal muscle dysfunction/wasting requires a better understanding of the molecular mechanisms responsible for these abnormalities. Microarrays are powerful tools that allow the investigation of the expression of thousands of genes, virtually the whole genome, simultaneously. We aim at identifying genes and molecular pathways involved in skeletal muscle wasting in COPD.MethodsWe assessed and compared the vastus lateralis transcriptome of COPD patients with low fat free mass index (FFMI) as a surrogate of muscle mass (COPDL) (FEV1 30 ± 3.6%pred, FFMI 15 ± 0.2 Kg.m−2) with patients with COPD and normal FFMI (COPDN) (FEV1 44 ± 5.8%pred, FFMI 19 ± 0.5 Kg.m−2) and a group of age and sex matched healthy controls (C) (FEV1 95 ± 3.9%pred, FFMI 20 ± 0.8 Kg.m−2) using Agilent Human Whole Genome 4x44K microarrays. The altered expression of several of these genes was confirmed by real time TaqMan PCR. Protein levels of P21 were assessed by immunoblotting.ResultsA subset of 42 genes was differentially expressed in COPDL in comparison to both COPDN and C (PFP < 0.05; −1.5 ≥ FC ≥ 1.5). The altered expression of several of these genes was confirmed by real time TaqMan PCR and correlated with different functional and structural muscle parameters. Five of these genes (CDKN1A, GADD45A, PMP22, BEX2, CGREF1, CYR61), were associated with cell cycle arrest and growth regulation and had been previously identified in studies relating muscle wasting and ageing. Protein levels of CDKN1A, a recognized marker of premature ageing/cell cycle arrest, were also found to be increased in COPDL.ConclusionsThis study provides evidence of differentially expressed genes in peripheral muscle in COPD patients corresponding to relevant biological processes associated with skeletal muscle wasting and provides potential targets for future therapeutic interventions to prevent loss of muscle function and mass in COPD.

Neuromuscular electrical stimulation prevents muscle function deterioration in exacerbated COPD: A pilot study

PURPOSE COPD is a condition with systemic effects of which peripheral muscle dysfunction is a prominent contributor to exercise limitation, health related quality of life (HRQoL) impairment, and is an independent predictor of morbidity and mortality. Pulmonary rehabilitation (PR) is a successful strategy to improve exercise tolerance and HRQoL through the improvement of muscle function in patients with stable COPD or early after severe exacerbations of COPD (SECOPD). However, muscle function further deteriorates during SECOPD before early PR programmes commence. We aimed to investigate the feasibility and efficacy of quadriceps neuromuscular electrical stimulation (NMES) applied during a SECOPD to prevent muscle function deterioration. METHODS We have conducted a pilot study in eleven COPD patients (FEV(1) 41.3 ± 5.6 % pred) admitted to hospital with a SECOPD. We randomly allocated one leg to receive NMES (once a day for 14 days) with the other leg as a control (non-stimulated leg). We measured the change in quadriceps maximal voluntary contraction (ΔQMVC) as the main outcome. RESULTS Mean quadriceps muscle strength decreased in control legs (ΔQMVC -2.9 ± 5.3 N, p = ns) but increased in the stimulated legs (ΔQMVC 19.2 ± 6.1 N, p < 0.01). The difference in ΔQMVC between groups was statistically significant (p < 0.05). The effect of NMES was directly related to the stimulation intensity (∑mA) applied throughout the 14 sessions (r = 0.76, p < 0.01). All patients tolerated NMES without any side effects. CONCLUSIONS NMES is a feasible and effective treatment to prevent quadriceps muscle strength derangement during severe exacerbations of COPD and may be used to compliment early post-exacerbation pulmonary rehabilitation.