Javier Milara

Epithelial to mesenchymal transition is increased in patients with COPD and induced by cigarette smoke

Background Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of remodelling, peribronchiolar fibrosis is observed in the small airways of patients with COPD and contributes to airway obstruction. Epithelial to mesenchymal transition (EMT) appears to be involved in the formation of peribronchiolar fibrosis. This study examines the EMT process in human bronchial epithelial cells (HBECs) from non-smokers, smokers and patients with COPD as well as the in vitro effect of cigarette smoke extract (CSE) on EMT. Methods HBECs from non-smokers (n=5), smokers (n=12) and patients with COPD (n=15) were collected to measure the mesenchymal markers α-smooth muscle actin, vimentin and collagen type I and the epithelial markers E-cadherin, ZO-1 and cytokeratin 5 and 18 by real time-PCR and protein array. In vitro differentiated bronchial epithelial cells were stimulated with CSE. Results Mesenchymal markers were upregulated in HBECs of smokers and patients with COPD compared with non-smokers. In contrast, epithelial cell markers were downregulated. In vitro differentiated HBECs underwent EMT after 72 h of CSE exposure through the activation of intracellular reactive oxygen species, the release and autocrine action of transforming growth factor β1, the phosphorylation of ERK1/2 and Smad3 and by the downregulation of cyclic monophosphate. Conclusions The EMT process is present in bronchial epithelial cells of the small bronchi of smokers and patients with COPD and is activated by cigarette smoke in vitro.

Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition

Background Idiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) have been implicated in lung myofibroblast transition, but their role in EMT and their expression in patients with IPF is unknown. Methods and results S1P levels were measured in serum (n=27) and bronchoalveolar lavage (BAL; n=15) from patients with IPF and controls (n=30 for serum and n=15 for BAL studies). SPHK1 expression was measured in lung tissue from patients with IPF (n=12) and controls (n=15). Alveolar type II transformation into mesenchymal cells was studied in response to S1P (10−9–10−5 M). The median (IQR) of S1P serum levels was increased in patients with IPF (1.4 (0.4) μM) versus controls (1 (0.26) μM; p<0.0001). BAL S1P levels were increased in patients with IPF (1.12 (0.53) μM) versus controls (0.2 (0.5); p<0.0001) and correlated with diffusion capacity of the lung for carbon monoxide, forced expiratory volume in 1 s and forced vital capacity (Spearmans r=−0.87, −0.72 and −0.68, respectively) in patients with IPF. SPHK1 was upregulated in lung tissue from patients with IPF and correlated with α-smooth muscle actin, vimentin and collagen type I (Spearmans r=0.82, 0.85 and 0.72, respectively). S1P induced EMT in alveolar type II cells by interacting with S1P2 and S1P3, as well as by the activation of p-Smad3, RhoA-GTP, oxidative stress and transforming growth factor-β1 (TGF-β1) release. Furthermore, TGF-β1-induced EMT was partially conducted by the S1P/SPHK1 activation, suggesting crosstalk between TGF-β1 and the S1P/SPHK1 axis. Conclusions S1P is elevated in patients with IPF, correlates with the lung function and mediates EMT.

Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease

BACKGROUND Glucocorticoid functions are markedly impaired in patients with chronic obstructive pulmonary disease (COPD). The phosphodiesterase 4 inhibitor roflumilast N-oxide (RNO) is the active metabolite of roflumilast approved as a treatment to reduce the risk of exacerbations in patients with severe COPD. OBJECTIVE We sought to characterize the differential effects of RNO versus corticosteroids and their potential additive/synergistic effect in neutrophils from patients with COPD, thus providing scientific rationale for the combination of roflumilast with corticosteroids in the clinic. METHODS Peripheral blood neutrophils were isolated from patients with COPD (n = 32), smokers (n = 7), and healthy nonsmokers (n = 25). Levels of IL-8, matrix metallopeptidase 9 (MMP-9), and biomarkers of glucocorticoid resistance were determined by using ELISA and RT-PCR. Neutrophils were incubated with dexamethasone (0.1 nmol/L to 1 μmol/L), RNO (0.1 nmol/L to 1 μmol/L), or the combination of 1 nmol/L RNO plus 10 nmol/L DEX and stimulated with LPS (1 μg/mL) or cigarette smoke extract 5%; levels of IL-8, MMP-9, and other biomarkers were measured at the end of the incubation period. RESULTS Peripheral neutrophils from patients with COPD showed a primed phenotype with an increased basal release of IL-8 and MMP-9 and expressed a corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase δ, macrophage migration inhibitory factor, and glucocorticoid receptor β expression and a decrease in HDAC activity and mitogen-activated protein kinase phosphatase 1 expression. RNO demonstrated robust anti-inflammatory effects on neutrophils from patients with COPD, reversing their resistance to corticosteroids. The combination of RNO and dexamethasone showed additive/synergistic effects, which were consistent with the reversal of corticosteroid-resistant molecular markers by RNO. CONCLUSION RNO reverses corticosteroid resistance and shows strong anti-inflammatory effects alone or in combination with corticosteroids on neutrophils from patients with COPD.

Tobacco, Inflammation, and Respiratory Tract Cancer

Cigarette smoking is the most recognized risk factor for many inflammatory diseases such as cardiovascular diseases, chronic obstructive pulmonary disease and for a number of malignances such as lung cancer. Lung cancer is currently considered the leading cause of cancer-related deaths because its aggressive nature and the lack of effective therapeutic options. Recent advances in molecular biology and immunology have improved the knowledge on different mechanisms implicated in lung cell malignant transformation, progression and metastasis, thus presenting an exciting new era for lung anticancer therapies. The way by which cigarette smoke may induce lung malignancy includes a large number of different mechanisms and substances, most of them currently unknown. Thus, identified carcinogenic compounds of cigarette smoke may induce themselves a direct cytotoxicity and mutagenic action on lung epithelial cells by means of generation of somatic mutations, epigenetic events, epithelial cell to mesenchymal cell transformations, as well as by chronic cell damage. However, the fact that there is a relative high prevalence of ex-smoker who may develop lung cancer after years of smoking cessation suggest that other causes are also implicated. Thus cigarette smoke-induced chronic lung inflammatory microenvironment, oxidative stress and cell structural alterations such as the increase of cell proliferation, angiogenesis and apoptosis arrest are irreversible processes that have a high influence in lung tumor growth. In this review we focused in current knowledge on the mechanisms implicated in cigarette smoke-induced lung chronic inflammatory processes leading to lung carcinogenesis, as well as in current therapies based on novel molecular advances.

Effects of long‐term azithromycin therapy on airway oxidative stress markers in non‐cystic fibrosis bronchiectasis

To explore the effect of long‐term therapy with azithromycin in regards to airway oxidative stress markers in exhaled breath condensate (EBC) of adult patients with stable non‐cystic fibrosis (CF) bronchiectasis.

Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil.

Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in the pathogenesis of several lung disorders. In this study we evaluated the effect of PDE5 inhibition on pulmonary artery endothelial dysfunction induced by cigarette smoke in vitro. Human pulmonary artery endothelial cells (HPAEC) were incubated in the absence or presence of PDE5 inhibitor sildenafil (10 nM-1 microM), PKG agonist 8-Br-cGMP (1mM), or the antioxidants dyphenyleneiodonium (DPI 1 microM) and N-acetylcysteine (NAC 1mM) for 30 min. Then, cigarette smoke extract (CSE) was added for 24h. CSE (2.5-10%)-induced ROS generation was suppressed by DPI, and partially reversed by sildenafil and 8-Br-cGMP. Decreases in intracellular levels of cGMP and extracellular NO induced by CSE were reversed by sildenafil and DPI. Furthermore, CSE-induced pg91(phox) and PDE5 mRNA overexpression were suppressed by both sildenafil and DPI. CSE (2.5-10%) induced upregulation of IL-6, IL-8 and Ang-2, and decreased Ang-1 expression in parallel to apoptosis which were partially suppressed by sildenafil, 8-Br-cGMP, DPI and NAC. This study demonstrates that PDE5 inhibition attenuates the oxidant burden and the inflammatory and remodeling effects of CSE in human HPAEC which may contribute to the therapeutic value of PDE5 inhibitors for pulmonary disorders coursing with endothelial dysfunction.

Oxidative stress-induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages

Background Oxidative stress is present in airway diseases such as severe asthma or Chronic Obstructive Pulmonary Disease and contributes to the low response to glucocorticoids through the down‐regulation of histone deacetylase (HDAC) activity.

Cigarette smoke exposure up‐regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium‐dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ETB) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

Neutrophil Activation in Severe, Early-Onset COPD Patients versus Healthy Non-Smoker Subjects in vitro: Effects of Antioxidant Therapy

Background: Neutrophils and oxidative stress have been implicated in the pathogenesis of COPD. Severe, early-onset COPD is characterized by a rapid decline in the lung function at an early age; however, nothing is known about neutrophil activation in COPD patients. Objectives: The aim of this study was to evaluate peripheral blood neutrophil activation in severe, early-onset COPD patients versus healthy non-smokers and the effect of N-acetyl-L-cysteine (NAC) on neutrophil activation in vitro. Methods: Neutrophils were isolated from 15 severe, early-onset COPD patients and 15 age-matched healthy subjects and stimulated with N-formyl- Met-Leu-Phe (fMLP) in the presence or absence of NAC (10 µM to 10 mM). Neutrophil chemotaxis, elastase release, reactive oxygen species (ROS), intracellular thiols and apoptosis were measured by Boyden chamber, spectrofluorometry, CMFDA and H2DCF-DA dyes and by annexin V-FITC, respectively. Results: Chemotaxis of peripheral blood neutrophils from COPD patients in response to fMLP was 30% more increased than that observed in healthy subjects. Elastase release in response to fMLP was 2-fold higher in neutrophils from COPD patients versus healthy subjects. Intracellular thiol levels were 30% lower in COPD and ROS was approximately 30% higher in COPD versus healthy neutrophils. Spontaneous apoptosis showed no differences in both groups of patients and fMLP-induced apoptosis was higher in COPD. Pre-treatment with the antioxidant NAC effectively attenuated neutrophil chemotaxis, elastase release and ROS as well as effectively increased thiol levels in COPD. Conclusions: Neutrophils in severe, early-onset COPD patients are highly activated and this is alleviated by NAC in vitro.

Crataegus laevigata decreases neutrophil elastase and has hypolipidemic effect: a randomized, double-blind, placebo-controlled trial.

Crataegus laevigata is a medicinal plant most commonly used for the treatment of heart failure and psychosomatic disorders. Based on previous experimental findings, this double-blind placebo-controlled study was aimed at finding beneficial effects of C. laevigata on biomarkers of coronary heart disease (CHD). The study included 49 diabetic subjects with chronic CHD who were randomly assigned to the treatment for 6 months with either a micronized flower and leaf preparation of C. laevigata (400 mg three times a day) or a matching placebo. Blood cell count, lipid profile, C-reactive protein, neutrophil elastase (NE) and malondialdehyde were analyzed in plasma at baseline, at one month and six months. The main results were that NE decreased in the C. laevigata group compared to the placebo group. In the C. laevigata group, baseline figures (median and interquartile range) were 35.8 (4.5) and in the placebo group 31 (5.9). At the end of the study, values were 33.2 (4.7) ng/ml and 36.7 (2.2) ng/ml, respectively; p<0.0001. C. laevigata, added to statins, decreased LDL cholesterol (LDL-C) (mean±SD) from 105±28.5 mg/dl at baseline to 92.7±25.1 mg/dl at 6 months (p=0.03), and non-HDL cholesterol from 131±37.5 mg/dl to 119.6±33 mg/dl (p<0.001). Differences between groups did not reach statistical significance at 6 months. No significant changes were observed in the rest of parameters. In conclusion, C. laevigata decreased NE and showed a trend to lower LDL-C compared to placebo as add-on-treatment for diabetic subjects with chronic CHD.