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Almudever P.,University of Valencia | Milara J.,University of Valencia | Milara J.,Polytechnic University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | And 13 more authors.
Thorax | Year: 2013

Background Pulmonary hypertension in idiopathic pulmonary fibrosis (IPF) is indicative of a poor prognosis. Recent evidence suggests that tetrahydrobiopterin (BH4), the cofactor of nitric oxide synthase (NOS), is involved in pulmonary hypertension and that pulmonary artery endothelial-to-mesenchymal transition (EnMT) may contribute to pulmonary fibrosis. However, the role of BH4 in pulmonary remodelling secondary to pulmonary fibrosis is unknown. This study examined the BH4 system in plasma and pulmonary arteries from patients with IPF as well as the antiremodelling and antifibrotic effects of the BH4 precursor sepiapterin in rat bleomycin-induced pulmonary fibrosis and in vitro EnMT models. Methods BH4 and nitrotyrosine were measured by high-performance liquid chromatography and ELISA, respectively. Expression of sepiapterin reductase (SPR), GTP cyclohydrolase 1 (GCH-1), endothelial NOS (eNOS) and inducible NOS (iNOS) were measured by quantitative PCR and immunohistochemistry. Results BH4 plasma levels were downregulated in patients with IPF compared with controls while nitrites, nitrates and nitrotyrosine were upregulated. GCH-1 and eNOS were absent in pulmonary arteries of patients with IPF; however, iNOS expression increased while SPR expression was unchanged. In rats, oral sepiapterin (10 mg/kg twice daily) attenuated bleomycin-induced pulmonary fibrosis, mortality, vascular remodelling and pulmonary hypertension by increasing rat plasma BH4, decreasing plasma nitrotyrosine and increasing vascular eNOS and GCH-1 expression. Both transforming growth factor β1 and endothelin-1 induced EnMT by decreasing BH4 and eNOS expression. In vitro administration of sepiapterin increased endothelial BH4 and inhibited EnMT in human pulmonary artery endothelial cells. Conclusions Targeting the BH4 synthesis 'salvage pathway' with sepiapterin may be a new therapeutic strategy to attenuate pulmonary hypertension in IPF.


PubMed | Foundation University, University of Valencia and Research Foundation of General Hospital of Valencia
Type: Journal Article | Journal: The Journal of allergy and clinical immunology | Year: 2015

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.To analyze the role of MUC1 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved.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.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 GR form protein complexes and translocate to the nucleus in response to dexamethasone. MUC1-CT-GR complex was downregulated in NP-CR tissue.MUC1-CT participates in the corticosteroid response that mediates GR nuclear translocation. The low expression of MUC1 in patients with CRSwNP may participate in corticosteroid resistance.


PubMed | Foundation University, University of Valencia, Research Foundation of General Hospital of Valencia and Takeda Pharmaceuticals International
Type: Journal Article | Journal: Pulmonary pharmacology & therapeutics | Year: 2014

Epithelial to mesenchymal transition (EMT) is under discussion as a potential mechanism of small airway remodelling in COPD. In bronchial epithelium of COPD and smokers markers of EMT were described. Invitro, EMT may be reproduced by exposing well-differentiated human bronchial epithelial cells (WD-HBEC) to cigarette smoke extract (CSE). EMT may be mitigated by an increase in cellular cAMP.This study explored the effects of roflumilast N-oxide, a PDE4 inhibitor on CSE-induced EMT in WD-HBEC and in primary bronchial epithelial cells from smokers and COPD invitro.WD-HBEC from normal donors were stimulated with CSE (2.5%) for 72h in presence of roflumilast N-oxide (2nM or 1M) or vehicle. mRNA and protein of EMT markers SMA, vimentin, collagen-1, E-cadherin, ZO-1, KRT5 as well as NOX4 were quantified by real-time quantitative PCR or protein array, respectively. Phosphorylated and total ERK1/2 and Smad3 were assessed by protein array. cAMP and TGF1 were measured by ELISA. Reactive oxygen species (ROS) were determined by DCF fluorescence, after 30min CSE (2.5%). Apoptosis was measured with Annexin V/PI labelling. In some experiments, EMT markers were determined in monolayers of bronchial epithelial cells from smokers, COPD versus controls.Roflumilast N-oxide protected from CSE-induced EMT in WD-HBEC. The PDE4 inhibitor reversed both the increase in mesenchymal and the loss in epithelial EMT markers. Roflumilast N-oxide restored the loss in cellular cAMP following CSE, reduced ROS, NOX4 expression, the increase in TGF1 release, phospho ERK1/2 and Smad3. The PDE4 inhibitor partly protected from the increment in apoptosis with CSE. Finally the PDE4 inhibitor decreased mesenchymal yet increased epithelial phenotype markers in HBEC of COPD and smokers.Roflumilast N-oxide may mitigate epithelial-mesenchymal transition in bronchial epithelial cells invitro.


Ortiz J.L.,University of Valencia | Milara J.,University of Valencia | Milara J.,Polytechnic University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | And 6 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2013

Background Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)-4 inhibitor. Methods Interleukin (IL)-8 was measured in cell supernatants by ELISA. Histone deacetylase (HDAC), histone acetylase (HAT), and intracellular cAMP levels were measured by colorimetric assays and enzyme immunoassay, respectively. PDE4 isotypes and glucocorticoid receptor (GR)-α and β expression were measured by real-time RT-PCR. Results The PDE4 inhibitor rolipram dose dependently inhibited the IL-8 secretion induced by CSE 5%. In contrast, dexamethasone 1 μM did not show inhibitory effect on IL-8 secretion. Combination of subeffective rolipram concentrations at 10 nM increased the inhibitory effect of dexamethasone to ~45% of inhibition. Cigarette smoke extract 5% inhibited HDAC activity and increased HAT activity generating glucocorticoid insensitivity. Rolipram did not modify the HDAC activity, however partially inhibited the increase in HAT activity at 1 μM. PDE4 isotypes were up-regulated by CSE 5% with the consequent cAMP down-regulation. Dexamethasone reduced all PDE4 isotypes expression and showed additive effects with rolipram enhancing cAMP levels. Furthermore, rolipram enhanced GR-α expression and inhibited the increase in GR-β induced by CSE. Conclusions Combination of rolipram and dexamethasone shows additive properties in HPAECs under glucocorticoid insensitive conditions. These results may be of potential value in future anti-inflammatory therapies using combination of PDE4 inhibitors and glucocorticoids. © 2012 John Wiley & Sons A/S.


Milara J.,University of Valencia | Milara J.,Health Institute Carlos III | Milara J.,Research Foundation of General Hospital of Valencia | Morcillo E.,Health Institute Carlos III | And 6 more authors.
PLoS ONE | Year: 2015

Fibrotic remodeling is a process common to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome and asthma. Based on preclinical studies phosphodiesterase 4 (PDE4) inhibitors may exhibit beneficial anti-inflammatory and anti-remodeling properties for the treatment of these respiratory disorders. Effects of PDE4 inhibitors on changes in the lung metabolome in models of pulmonary fibrotic remodeling have not yet been explored. This work studies the effects of the PDE4 inhibitor roflumilast on changes in the lung metabolome in the common murine model of bleomycin-induced lung fibrosis by nuclear magnetic resonance (NMR) metabolic profiling of intact lung tissue. Metabolic profiling reveals strong differences between fibrotic and non-fibrotic tissue. These differences include increases in proline, glycine, lactate, taurine, phosphocholine and total glutathione and decreases in global fatty acids. In parallel, there was a loss in plasma BH4. This profile suggests that bleomycin produces alterations in the oxidative equilibrium, a strong inflammatory response and activation of the collagen synthesis among others. Roflumilast prevented most of these metabolic effects associated to pulmonary fibrosis suggesting a favorable anti-fibrotic profile. Copyright: © 2015 Milara et al.


Milara J.,University of Valencia | Milara J.,Polytechnic University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | Peiro T.,Research Foundation of General Hospital of Valencia | And 9 more authors.
Pulmonary Pharmacology and Therapeutics | Year: 2014

Background: Epithelial to mesenchymal transition (EMT) is under discussion as a potential mechanism of small airway remodelling in COPD. In bronchial epithelium of COPD and smokers markers of EMT were described. Invitro, EMT may be reproduced by exposing well-differentiated human bronchial epithelial cells (WD-HBEC) to cigarette smoke extract (CSE). EMT may be mitigated by an increase in cellular cAMP. Objective: This study explored the effects of roflumilast N-oxide, a PDE4 inhibitor on CSE-induced EMT in WD-HBEC and in primary bronchial epithelial cells from smokers and COPD invitro. Methods: WD-HBEC from normal donors were stimulated with CSE (2.5%) for 72h in presence of roflumilast N-oxide (2nM or 1μM) or vehicle. mRNA and protein of EMT markers αSMA, vimentin, collagen-1, E-cadherin, ZO-1, KRT5 as well as NOX4 were quantified by real-time quantitative PCR or protein array, respectively. Phosphorylated and total ERK1/2 and Smad3 were assessed by protein array. cAMP and TGFβ1 were measured by ELISA. Reactive oxygen species (ROS) were determined by DCF fluorescence, after 30min CSE (2.5%). Apoptosis was measured with Annexin V/PI labelling. In some experiments, EMT markers were determined in monolayers of bronchial epithelial cells from smokers, COPD versus controls. Results: Roflumilast N-oxide protected from CSE-induced EMT in WD-HBEC. The PDE4 inhibitor reversed both the increase in mesenchymal and the loss in epithelial EMT markers. Roflumilast N-oxide restored the loss in cellular cAMP following CSE, reduced ROS, NOX4 expression, the increase in TGFβ1 release, phospho ERK1/2 and Smad3. The PDE4 inhibitor partly protected from the increment in apoptosis with CSE. Finally the PDE4 inhibitor decreased mesenchymal yet increased epithelial phenotype markers in HBEC of COPD and smokers. Conclusions: Roflumilast N-oxide may mitigate epithelial-mesenchymal transition in bronchial epithelial cells invitro. © 2014 Elsevier Ltd.


Milara J.,University of Valencia | Milara J.,Polytechnic University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | Peiro T.,Research Foundation of General Hospital of Valencia | And 10 more authors.
Journal of Allergy and Clinical Immunology | Year: 2014

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. © 2014 American Academy of Allergy, Asthma & Immunology.


Milara J.,University of Valencia | Milara J.,Polytechnic University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | Lluch J.,Research Foundation of General Hospital of Valencia | And 8 more authors.
Journal of Allergy and Clinical Immunology | Year: 2014

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. © 2014 American Academy of Allergy, Asthma and Immunology.


Pastor-Clerigues A.,University of Valencia | Marti-Bonmati E.,University of Valencia | Milara J.,University of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | And 5 more authors.
PLoS ONE | Year: 2014

Home parenteral nutrition (PN) is associated with many complications including severe hepatobiliary dysfunction. Commercial ω-6 fatty acid-soybean based-lipid emulsions in PN may mediate long term PN associate liver disease (PNALD) whereas ω-3-fish oil parenteral emulsions have shown to reverse PNALD in children. However, its clinical effectiveness in adults has been scarcely reported. In this work, we study the role of soybean and fish oil lipid commercial emulsions on inflammatory and profibrotic liver markers in adults with long term PNALD and in in vitro cellular models. Inflammatory and profibrotic markers were measured in serum of ten adults with long term PNALD and in culture supernatants of monocytes. Liver epithelial to mesenchymal transition (EMT) was induced by transforming growth factor beta 1 (TGFβ1) to evaluate in vitro liver fibrosis. Omegaven®, a 100% fish oil commercial emulsion, was infused during four months in two patients with severe long term PNALD reversing, at the first month, the inflammatory, profibrotic and clinical parameters of PNALD. The effect was maintained during the treatment course but impaired when conventional lipid emulsions were reintroduced. The other patients under chronic soybean oil-based PN showed elevated inflammatory and profibrotic parameters. In vitro human monocytes stimulated with lipopolysaccharide induced a strong inflammatory response that was suppressed by Omegaven®, but increased by soybean emulsions. In other experiments, TGFβ1 induced EMT that was suppressed by Omegaven® and enhanced by soybean oil lipid emulsions. Omegaven® improves clinical, antiinflammatory and anti-fibrotic parameters in adults with long-term home PNALD. © 2014 Pastor-Clerigues et al.


Milara J.,University of Valencia | Milara J.,General Hospital of Valencia | Milara J.,Research Foundation of General Hospital of Valencia | Milara J.,General Universitario | And 11 more authors.
COPD: Journal of Chronic Obstructive Pulmonary Disease | Year: 2015

Cigarette smoking contributes to epithelial-mesenchymal transition (EMT) in COPD small bronchi as part of the lung remodeling process. We recently observed that roflumilast N-oxide (RNO), the active metabolite of the PDE4 inhibitor roflumilast, prevents cigarette smoke-induced EMT in differentiated human bronchial epithelial cells. Further, statins were shown to protect renal and alveolar epithelial cells from EMT. Objectives: To analyze how RNO and simvastatin (SIM) interact on CSE-induced EMT in well-differentiated human bronchial epithelial cells (WD-HBEC) from small bronchi in vitro. Methods: WD-HBEC were stimulated with CSE (2.5%). The mesenchymal markers vimentin, collagen type I and α-SMA, the epithelial markers E-cadherin and ZO-1, as well as β-catenin were quantified by real time quantitative PCR or Western blotting. Intracellular reactive oxygen species (ROS) were measured using the H2DCF-DA probe. GTP-Rac1 and pAkt were evaluated by Western blotting. Results: The combination of RNO at 2 nM and SIM at 100 nM was (over) additive to reverse CSE-induced EMT. CSE-induced EMT was partially mediated by the generation of ROS and the activation of the PI3K/Akt/β-catenin pathway. Both RNO at 2 nM and SIM at 100 nM partially abrogated this pathway, and its combination almost abolished ROS/PI3K/Akt/β-catenin signaling and therefore EMT. Conclusions: The PDE4 inhibitor roflumilast N-oxide acts (over)additively with simvastatin to prevent CSE-induced EMT in WD-HBEC in vitro. © 2015 Informa Healthcare USA, Inc.

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