Adela Serrano

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.

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.

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.

Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps

BACKGROUND A number of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to oral corticosteroids. Mucin 1 (MUC1) shows anti-inflammatory properties, and its cytoplasmic tail (CT) interacts with transcription factors, facilitating their nuclear translocation. Because glucocorticoid receptor (GR) nuclear translocation is key to the anti-inflammatory effect of corticosteroids, we hypothesized that MUC1 is involved in the effectiveness of corticosteroids. OBJECTIVE To analyze the role of MUC1 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. METHODS Seventy-three patients with CRSwNP took oral corticosteroids for 15 days. Corticosteroid resistance was evaluated by nasal endoscopy. The expression of MUC1 and MUC1 CT was evaluated by real-time PCR, Western blotting, and immunohistochemistry. Beas-2B knockdown with RNA interference for MUC1 (siRNA-MUC1) was used to analyze the role of MUC1 in the anti-inflammatory effects of dexamethasone. RESULTS Nineteen patients had nasal polyps that were resistant to oral corticosteroids (NP-CR). MUC1 expression was downregulated in these patients. Primary epithelial cells from patients with NP-CR were insensitive to the anti-inflammatory effects of dexamethasone. In siRNA-MUC1 Beas-2B, dexamethasone showed weaker anti-inflammatory effects, a reduced inhibition of phospho-extracellular-signal-regulated kinases 1/2, a less severe mitogen-activated protein kinase phosphatase 1 increase, and a reduced GR nuclear translocation. Immunoprecipitation experiments revealed that MUC1-CT and GRα form protein complexes and translocate to the nucleus in response to dexamethasone. MUC1-CT-GRα complex was downregulated in NP-CR tissue. CONCLUSION MUC1-CT participates in the corticosteroid response that mediates GRα nuclear translocation. The low expression of MUC1 in patients with CRSwNP may participate in corticosteroid resistance.

Aclidinium inhibits cigarette smoke-induced lung fibroblast-to-myofibroblast transition

Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of this remodelling, peribronchiolar fibrosis is observed in the small airways of COPD patients and contributes to airway obstruction. Fibroblast-to-myofibroblast transition is a key step in peribronchiolar fibrosis formation. This in vitro study examined the effect of cigarette smoke on bronchial fibroblast-to-myofibroblast transition, and whether aclidinium bromide inhibits this process. Human bronchial fibroblasts were incubated with aclidinium bromide (10−9–10−7 M) and exposed to cigarette smoke extract. Collagen type I and &agr;-smooth muscle actin (&agr;-SMA) expression were measured by real-time PCR and Western blotting, as myofibroblast markers. Intracellular reactive oxygen species, cyclic AMP (cAMP), extracellular signal-regulated kinase (ERK)1/2 and choline acetyltransferase were measured as intracellular signalling mediators. Cigarette smoke-induced collagen type I and &agr;-SMA was mediated by the production of reactive oxygen species, the depletion of intracellular cAMP and the increase of ERK1/2 phosphorylation and choline acetyltransferase. These effects could be reversed by treatment with the anticholinergic aclidinium bromide, by silencing the mRNA of muscarinic receptors M1, M2 or M3, or by the depletion of extracellular acetylcholine by treatment with acetylcholinesterase. A non-neuronal cholinergic system is implicated in cigarette smoke-induced bronchial fibroblast-to-myofibroblast transition, which is inhibited by aclidinium bromide.

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair

The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca(2+) ([Ca(2+)](out)) which resulted in a concentration-dependent intracellular Ca(2+) ([Ca(2+)](i)) increase (potency approximately 5.6mM [Ca(2+)](out)). Furthermore, increasing [Ca(2+)](out) induced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) which was blocked by siRNA-CaSR and the specific inhibitor of CaSR, NPS2390. Epithelial repair after mechanical injury of differentiated HBEC was a process dependent of [Ca(2+)](out) since it accelerated wound repair and HBEC proliferation being highest at 5mM [Ca(2+)](out). Furthermore, U73122 (an inhibitor of phospholipase C (PLC)) and PD 98059 (an inhibitor of ERK1/2) as well as siRNA-CaSR and NPS2390 partially inhibited wound repair and HBEC proliferation. On the other hand, mechanical injury produced an [Ca(2+)](i) wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca(2+) chelator EGTA, which suggest a link of CaSR between cell-cell communication and wound repair in differentiated HBEC. Our data, for the first time, shows that CaSR plays an important role in airway epithelial repair, which may help to develop novel regenerative therapeutics allowing the rapid repair of lung damaged epithelium.

Evaluation of the Ocular Tolerance of Three Tacrolimus Topical Pharmaceutical Preparations by Bovine Corneal Opacity and Permeability Test

ABSTRACT Purpose: Tacrolimus ocular preparations are commonly employed in autoimmune or inflammatory ocular disorders. However, currently there are not yet approved ocular formulations. Tacrolimus ocular side effects have been reported in clinical use, so the evaluation of different pharmaceutical preparations is mandatory. In this study, the local corneal tolerance and safety profile of three common tacrolimus 0.03% pharmaceutical preparations were evaluated. Material and methods: Corneal irritation and permeability of tacrolimus preparations were evaluated with the bovine corneal opacity and permeability (BCOP) test. Complementary corneal hematoxylin/eosin and immunohistochemistry staining for tight junctions and adherent junctions E-cadherin, VE-cadherin and zonula occludens-1 were examined and scored to evaluate and to confirm corneal disruption and irritation scores obtained with the BCOP method. Results: Commercial brand ointment (Protopic®), topical compounded eye ointment (pharmacy elaboration) and tacrolimus suspension eye drops (elaborated from parenteral prograf®) were tested as potential ocular preparations to be used in clinics. Tacrolimus preparations hereby studied do not alter the opacity and permeability of the bovine cornea by more than three units, measured by the In Vitro Irritancy Score, neither affected the immunohistochemical parameters, composite score or transepithelial electrical resistance. Conclusions: Tacrolimus preparations studied can be safely applied as a topical ocular treatment.

Bafetinib inhibits functional responses of human eosinophils in vitro.

Eosinophils play a prominent role in the process of allergic inflammation. Non-receptor associated Lyn tyrosine kinases generate key initial signals in eosinophils. Bafetinib, a specific Abl/Lyn tyrosine kinase inhibitor has shown a potent antiproliferative activity in leukemic cells, but its effects on eosinophils have not been reported. Therefore, we studied the effects of bafetinib on functional and mechanistic responses of isolated human eosinophils. Bafetinib was more potent than non-specific tyrosin kinase comparators genistein and tyrphostin inhibiting superoxide anion triggered by N-formyl-Met-Leu-Phe (fMLF; 100 nM) (-log IC50=7.25 ± 0.04 M; 6.1 ± 0.04 M; and 6.55 ± 0.03 M, respectively). Bafetinib, genistein and tyrphostin did not modify the [Ca(2+)]i responses to fMLF. Bafetinib inhibited the release of EPO induced by fMLF with higher potency than genistein and tyrphostin (-log IC50=7.24 ± 0.09 M; 5.36 ± 0.28 M; and 5.37 ± 0.19 M, respectively), and nearly suppressed LTC4, ECP and chemotaxis. Bafetinib, genistein and tyrphostin did not change constitutive apoptosis. However bafetinib inhibited the ability of granulocyte-monocyte colony-stimulating factor to prevent apoptosis. The activation of Lyn tyrosine kinase, p-ERK1/2 and p-38 induced by fMLF was suppressed by bafetinib and attenuated by genistein and tyrphostin. In conclusion, bafetinib inhibits oxidative burst and generation of inflammatory mediators, and reverses the eosinophil survival. Therefore, future anti-allergic therapies based on bafetinib, could help to suppress excessive inflammatory response of eosinophils at inflammatory sites.

Authors’ response to: Epithelial mesenchymal transition (EMT) in small airways of COPD patient

We understand the reservations highlighted by Sohal and Walters in their letter in response to our recent work published.1 In their letter, Sohal and Walters argue that E-cadherin and ZO-1 are absent in our immunohistochemistry analysis of small bronchi of smokers and COPD patients, and that if disappeared completely epithelium would fall apart.1 In our study, we detected downregulation of E-cadherin and ZO-1 as well as a change of intercellular and apical distribution to diffuse cytoplasmic redistribution (figure 3B; in our work published in Thorax on 7 January 2013),2 but not …