European Journal of Pharmaceutical Sciences, Mar 1, 2010
Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in ... more 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.
Background: Idiopathic pulmonary fibrosis (IPF) is characterized by a rapid progressive lung decl... more Background: Idiopathic pulmonary fibrosis (IPF) is characterized by a rapid progressive lung decline and premature death after its diagnosis. Roflumilast displayed anti-fibrotic effects in animal and cellular models. Recent studies indicate that the combination of PDE4 and PDE5 inhibitors (sildenafil) potentiates anti-fibrotic properties of each drug, suggesting potential beneficts of this combination. Objectives: To study the effects from adding sildenafil to roflumilast N-oxide (RNO) inhibiting TGFβ1-induced human alveolar type II (AECII) epithelial-to-mesenchymal transition (EMT) and human fibrocyte to myofibroblast transition in vitro. Methods: AECII and fibrocytes were isolated from healthy donors. Cells were pre-incubated with therapeutical concentrations of RNO (2nM) and/or sildenafil (10nM) and stimulated with TGFβ1 for 72h. Studies on chemotaxis, cell viability (annexin V), different markers of EMT and myofibroblast were performed. Results: RNO/sildenafil combination significantly reduced TGFβ1- induced gene and protein expression of mesenchymal/myofibroblast markers collagen tye I, alpha actin smooth muscle and vimentin, prevented the loss of epitelial markers E-cadherin and ZO-1 and inhibited the change of cell phenotype observed in the EMT. Inhibition of fibrocyte to myofibroblast transition and fibrocyte chemotaxis was also potentiated by RNO/sildenafil combination. Annexin V studies showed that combination improved survival of AECII. RNO/sildenafil combination showed synergic properties. Conclusions: RNO/sildenafil combination show synergic activity, reduces mesenchymal markers expression and reverses EMT, improving anti-fibrotic effects in human AECII and fibrocytes.
Background: Corticoid resistance is relevant because implies a lower anti-inflammatory activity o... more Background: Corticoid resistance is relevant because implies a lower anti-inflammatory activity of corticoids. Diseases such as Chronic Rhinosinusitis with Nasal Polyposis (CRSwNP) may have corticoid resistance, while Chronic Obstructive Pulmonary Disease (COPD) is itself. Mucin 1 (MUC1) shows anti-inflammatory properties, and its cytoplasmic tail (CT) modulates transcription factors. Anti-inflammatory effect of corticoids requires glucocorticoid receptor (GR) nuclear translocation, then we hypothesize that MUC1 could modulate this mechanism Objective: To determine the role of MUC1 in corticoid effectiveness in CRSwNP and COPD Methods: The expression of MUC1 and MUC1-CT was determined in CRSwNP polyps and healthy or COPD lung parenchyma using RT-PCR, Western blotting and Immunohistochemistry. Lack of MUC1 with RNA interference (siRNA-MUC1) in BEAS -2B cell line was used to estimate its importance in the anti-inflammatory activity of dexamethasone Results: MUC1 expression was reduced in nasal polyps that were resistant to oral corticoids (NP-CR) and in lung parenchyma from smokers and COPD patients. NP-CR derived primary epithelial cells showed lack of the anti-inflammatory effects of dexamethasone. In siRNA-MUC1 Beas-2B, dexamethasone had weaker anti-inflammatory effects. Immunoprecipitation revealed that MUC1-CT and GRα interact and translocate to the nucleus in response to dexamethasone. MUC1-CT-GRα complex was reduced in NP-CR tissue Conclusion: Corticoid response that mediates GRα nuclear translocation requires MUC1-CT. The low expression of MUC1 in patients with CRSwNP or COPD may participate in corticoid resistance.
Background: Some patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to... more Background: Some patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to corticoids. Mucin 4 (MUC4) is a membrane anchored protein with a nuclear translocation domain which is modulated by corticoids. Because glucocorticoid receptor (GR) nuclear translocation is key to the anti-inflammatory effect of corticoids, we hypothesized that MUC4 is involved in the effectiveness of these drugs Objective: To analyze the role of MUC4 in corticoid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved Methods: 73 patients with CRSwNP took oral corticoids for 15 days. Corticoid resistance was evaluated by nasal endoscopy. The expression of MUC4 was determined by real-time PCR, Western blotting, and immunohistochemistry. Beas-2B knockdown with RNA interference for MUC4 (siRNA-MUC4) was used to analyze the role of MUC4 in the anti-inflammatory effects of dexamethasone Results: 90 patients had nasal polyps with corticoid resistance (NP-CR). MUC4 expression was upregulated in these patients. Primary epithelial cells from NP-CR were insensitive to the anti-inflammatory effects of dexamethasone. In siRNA-MUC4 Beas-2B, dexamethasone showed higher anti-inflammatory effects. Immunoprecipitation revealed that MUC4 and GRα form protein complexes modulated by dexamethasone. In epithelial cells from NP-CR MUC4-GRα complex is not dissociated in presence of dexamethasone, avoiding the GRα nuclear translocation and therefore its anti-inflammatory effects Conclusion: MUC4 participates in the corticoid response that mediates GRα nuclear translocation. The high expression of MUC4 in patients with CRSwNP may participate in corticoid resistance.
Background: Severe, early-onset COPD (Chronic Obstructive Pulmonary Disease) is characterized by ... more Background: Severe, early-onset COPD (Chronic Obstructive Pulmonary Disease) is characterized by a rapid decline in lung function at an early age with neutrophil over-activation. Roflumilast is approved as treatment for moderate and severe COPD at risk of exacerbation as add on therapy. Recent evidence indicates that the combination of PDE4 and PI3Kδ inhibitors show synergic anti-inflammatory properties. Objectives: To explore the effects from adding a selective PI3Kδ inhibitor to roflumilast N-oxide (RNO) in neutrophils isolated from peripheral blood of severe, early-onset COPD patients in in vitro models. Methods: Neutrophils were isolated from peripheral blood of 20 severe, early-onset COPD patients and stimulated with fMLP. Neutrophils were pre-incubated with RNO (0.1nM-1µM) and PI3Kδ inhibitor (5nM and 50nM) alone or combination. Chemotaxis, superoxide anion generation, neutrophil elastase and IL-8 releases were. measured. Results: RNO at 100nM and 1µM (but not at lower concentrations) significantly inhibited fMLP-induced superoxide anion generation, chemotaxis, IL-8 and elastase released by neutrophils isolated from early-onset COPD. PI3Kδ inhibitor at 50nM significantly inhibited fMLP-induced the inflammatory parameters involved in COPD. The combination of RNO (0.1nM) and PI3Kδ inhibitor (5nM) at non-effective concentrations showed synergic properties inhibiting neutrophil activation parameters in early-onset COPD patients. Conclusions: The combination of RNO with PI3kδ inhibitor show synergic activity inhibiting the characteristic “overactive” neutrophil phenotype of early-onset COPD patients.
Background: Corticosteroid resistance is an acquired condition in chronic obstructive pulmonary d... more Background: Corticosteroid resistance is an acquired condition in chronic obstructive pulmonary disease (COPD) patients and a challenge to develop new anti-inflammatory therapies. In previous reports we showed that cytoplasmic tail of the membrain tethered mucin 1 (MUC1-CT) interacts with glucocorticoid receptor (GR) mediating corticosteroid anti-inflammatory efficacy. Objectives: To analyze the role of MUC1-CT as a key marker of corticosteroid efficacy in COPD Methods: The expression of MUC1-CT and the anti-inflammatory role of dexamethasone were evaluated in neutrophils and bronchial epithelial cells from healthy and COPD patients. Anti-inflammatory effects of dexamethasone and glucocorticoid response element (GRE) activation were tested in bronchial epithelial cells silenced for MUC1 (siRNA-MUC1). Anti-inflammatory effects of dexamethasone were analyzed in a KO-MUC1 model of seven-day smoking mouse. Results: MUC1-CT was down-regulated in lung tissue, neutrophils and airway epithelial cells from COPD patients. Cigarette smoke extract down-regulated MUC1-CT expression and impaired dexamethasone anti-inflammatory effects. In siRNA-MUC1 cells, dexamethasone decreased its anti-inflammatory properties, and showed a lesser activation of GRE signal and induction of anti-inflammatory genes. Confocal fluorescence microscope analysis and immunoprecipitation experiments showed colocalization and interaction of MUC1-CT and GR in response to dexamethasone that decreased in presence of cigarette smoke. In KO-MUC1 smoker mouse model dexamethasone showed poor anti-inflammatory effects. Conclusions: MUC1-CT mediates the anti-inflammatory properties of corticosteroids and the lack of its expression in COPD increases resistance to corticosteroids.
INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is the interstitial pulmonary disease with highe... more INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is the interstitial pulmonary disease with higher incidence and worse prognosis. Recent evidence suggests that cucurbitaceae are selective inhibitors of the JAK2/STAT3 pathway. Since P-STAT3 is increased in fibrotic foci of IPF patients, we hypothesis that it may inhibit the progression of the disease. OBJECTIVE The purpose of this study was to evaluate the effect of Cucurbitacin I (CuI) in a murine model of bleomycin-induced pulmonary fibrosis. MATERIAL Y METHODS Wistar rats were instilled intratracheally with a single dose of bleomycin (BLM)(3.75 U/kg; n=12) to induce lung fibrosis. CuI (20mg/kg/day; n=6) or CuI vehicle (control and IPF group) was administered intraperitoneally daily for 21 days. Animal evolution was controlled through CT/SPECT imaging. Tgf-β1, Col1A and Et-1 gene and protein expression was measured by real time PCR and western blott as fibrotic markers. RESULTS CT/SPECT quantification showed reduction of the fibrotic areas in the CuI treated group by reestablishing the air space in the lungs by inhibiting the IPF progression. Tgf-β1, Col1A and Et-1 gene expression was upregulated 2.3, 7.2 and 1.9 fold respectively in BLM relative to Control rats. This was counteracted with CuI treatment. These results were substantiated by protein over-expression of Tgf-β1 (62.30%), Col1A (45.80%) and Et-1 (44.44%) in BLM rat. Protein expression was inhibited in CuI treated group by 32.04%, 30.52% and 44.99% respectively. CONCLUSION CuI inhibits the rat model BLM induced pulmonary fibrosis.
European Journal of Pharmaceutical Sciences, Mar 1, 2010
Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in ... more 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.
Background: Idiopathic pulmonary fibrosis (IPF) is characterized by a rapid progressive lung decl... more Background: Idiopathic pulmonary fibrosis (IPF) is characterized by a rapid progressive lung decline and premature death after its diagnosis. Roflumilast displayed anti-fibrotic effects in animal and cellular models. Recent studies indicate that the combination of PDE4 and PDE5 inhibitors (sildenafil) potentiates anti-fibrotic properties of each drug, suggesting potential beneficts of this combination. Objectives: To study the effects from adding sildenafil to roflumilast N-oxide (RNO) inhibiting TGFβ1-induced human alveolar type II (AECII) epithelial-to-mesenchymal transition (EMT) and human fibrocyte to myofibroblast transition in vitro. Methods: AECII and fibrocytes were isolated from healthy donors. Cells were pre-incubated with therapeutical concentrations of RNO (2nM) and/or sildenafil (10nM) and stimulated with TGFβ1 for 72h. Studies on chemotaxis, cell viability (annexin V), different markers of EMT and myofibroblast were performed. Results: RNO/sildenafil combination significantly reduced TGFβ1- induced gene and protein expression of mesenchymal/myofibroblast markers collagen tye I, alpha actin smooth muscle and vimentin, prevented the loss of epitelial markers E-cadherin and ZO-1 and inhibited the change of cell phenotype observed in the EMT. Inhibition of fibrocyte to myofibroblast transition and fibrocyte chemotaxis was also potentiated by RNO/sildenafil combination. Annexin V studies showed that combination improved survival of AECII. RNO/sildenafil combination showed synergic properties. Conclusions: RNO/sildenafil combination show synergic activity, reduces mesenchymal markers expression and reverses EMT, improving anti-fibrotic effects in human AECII and fibrocytes.
Background: Corticoid resistance is relevant because implies a lower anti-inflammatory activity o... more Background: Corticoid resistance is relevant because implies a lower anti-inflammatory activity of corticoids. Diseases such as Chronic Rhinosinusitis with Nasal Polyposis (CRSwNP) may have corticoid resistance, while Chronic Obstructive Pulmonary Disease (COPD) is itself. Mucin 1 (MUC1) shows anti-inflammatory properties, and its cytoplasmic tail (CT) modulates transcription factors. Anti-inflammatory effect of corticoids requires glucocorticoid receptor (GR) nuclear translocation, then we hypothesize that MUC1 could modulate this mechanism Objective: To determine the role of MUC1 in corticoid effectiveness in CRSwNP and COPD Methods: The expression of MUC1 and MUC1-CT was determined in CRSwNP polyps and healthy or COPD lung parenchyma using RT-PCR, Western blotting and Immunohistochemistry. Lack of MUC1 with RNA interference (siRNA-MUC1) in BEAS -2B cell line was used to estimate its importance in the anti-inflammatory activity of dexamethasone Results: MUC1 expression was reduced in nasal polyps that were resistant to oral corticoids (NP-CR) and in lung parenchyma from smokers and COPD patients. NP-CR derived primary epithelial cells showed lack of the anti-inflammatory effects of dexamethasone. In siRNA-MUC1 Beas-2B, dexamethasone had weaker anti-inflammatory effects. Immunoprecipitation revealed that MUC1-CT and GRα interact and translocate to the nucleus in response to dexamethasone. MUC1-CT-GRα complex was reduced in NP-CR tissue Conclusion: Corticoid response that mediates GRα nuclear translocation requires MUC1-CT. The low expression of MUC1 in patients with CRSwNP or COPD may participate in corticoid resistance.
Background: Some patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to... more Background: Some patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to corticoids. Mucin 4 (MUC4) is a membrane anchored protein with a nuclear translocation domain which is modulated by corticoids. Because glucocorticoid receptor (GR) nuclear translocation is key to the anti-inflammatory effect of corticoids, we hypothesized that MUC4 is involved in the effectiveness of these drugs Objective: To analyze the role of MUC4 in corticoid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved Methods: 73 patients with CRSwNP took oral corticoids for 15 days. Corticoid resistance was evaluated by nasal endoscopy. The expression of MUC4 was determined by real-time PCR, Western blotting, and immunohistochemistry. Beas-2B knockdown with RNA interference for MUC4 (siRNA-MUC4) was used to analyze the role of MUC4 in the anti-inflammatory effects of dexamethasone Results: 90 patients had nasal polyps with corticoid resistance (NP-CR). MUC4 expression was upregulated in these patients. Primary epithelial cells from NP-CR were insensitive to the anti-inflammatory effects of dexamethasone. In siRNA-MUC4 Beas-2B, dexamethasone showed higher anti-inflammatory effects. Immunoprecipitation revealed that MUC4 and GRα form protein complexes modulated by dexamethasone. In epithelial cells from NP-CR MUC4-GRα complex is not dissociated in presence of dexamethasone, avoiding the GRα nuclear translocation and therefore its anti-inflammatory effects Conclusion: MUC4 participates in the corticoid response that mediates GRα nuclear translocation. The high expression of MUC4 in patients with CRSwNP may participate in corticoid resistance.
Background: Severe, early-onset COPD (Chronic Obstructive Pulmonary Disease) is characterized by ... more Background: Severe, early-onset COPD (Chronic Obstructive Pulmonary Disease) is characterized by a rapid decline in lung function at an early age with neutrophil over-activation. Roflumilast is approved as treatment for moderate and severe COPD at risk of exacerbation as add on therapy. Recent evidence indicates that the combination of PDE4 and PI3Kδ inhibitors show synergic anti-inflammatory properties. Objectives: To explore the effects from adding a selective PI3Kδ inhibitor to roflumilast N-oxide (RNO) in neutrophils isolated from peripheral blood of severe, early-onset COPD patients in in vitro models. Methods: Neutrophils were isolated from peripheral blood of 20 severe, early-onset COPD patients and stimulated with fMLP. Neutrophils were pre-incubated with RNO (0.1nM-1µM) and PI3Kδ inhibitor (5nM and 50nM) alone or combination. Chemotaxis, superoxide anion generation, neutrophil elastase and IL-8 releases were. measured. Results: RNO at 100nM and 1µM (but not at lower concentrations) significantly inhibited fMLP-induced superoxide anion generation, chemotaxis, IL-8 and elastase released by neutrophils isolated from early-onset COPD. PI3Kδ inhibitor at 50nM significantly inhibited fMLP-induced the inflammatory parameters involved in COPD. The combination of RNO (0.1nM) and PI3Kδ inhibitor (5nM) at non-effective concentrations showed synergic properties inhibiting neutrophil activation parameters in early-onset COPD patients. Conclusions: The combination of RNO with PI3kδ inhibitor show synergic activity inhibiting the characteristic “overactive” neutrophil phenotype of early-onset COPD patients.
Background: Corticosteroid resistance is an acquired condition in chronic obstructive pulmonary d... more Background: Corticosteroid resistance is an acquired condition in chronic obstructive pulmonary disease (COPD) patients and a challenge to develop new anti-inflammatory therapies. In previous reports we showed that cytoplasmic tail of the membrain tethered mucin 1 (MUC1-CT) interacts with glucocorticoid receptor (GR) mediating corticosteroid anti-inflammatory efficacy. Objectives: To analyze the role of MUC1-CT as a key marker of corticosteroid efficacy in COPD Methods: The expression of MUC1-CT and the anti-inflammatory role of dexamethasone were evaluated in neutrophils and bronchial epithelial cells from healthy and COPD patients. Anti-inflammatory effects of dexamethasone and glucocorticoid response element (GRE) activation were tested in bronchial epithelial cells silenced for MUC1 (siRNA-MUC1). Anti-inflammatory effects of dexamethasone were analyzed in a KO-MUC1 model of seven-day smoking mouse. Results: MUC1-CT was down-regulated in lung tissue, neutrophils and airway epithelial cells from COPD patients. Cigarette smoke extract down-regulated MUC1-CT expression and impaired dexamethasone anti-inflammatory effects. In siRNA-MUC1 cells, dexamethasone decreased its anti-inflammatory properties, and showed a lesser activation of GRE signal and induction of anti-inflammatory genes. Confocal fluorescence microscope analysis and immunoprecipitation experiments showed colocalization and interaction of MUC1-CT and GR in response to dexamethasone that decreased in presence of cigarette smoke. In KO-MUC1 smoker mouse model dexamethasone showed poor anti-inflammatory effects. Conclusions: MUC1-CT mediates the anti-inflammatory properties of corticosteroids and the lack of its expression in COPD increases resistance to corticosteroids.
INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is the interstitial pulmonary disease with highe... more INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is the interstitial pulmonary disease with higher incidence and worse prognosis. Recent evidence suggests that cucurbitaceae are selective inhibitors of the JAK2/STAT3 pathway. Since P-STAT3 is increased in fibrotic foci of IPF patients, we hypothesis that it may inhibit the progression of the disease. OBJECTIVE The purpose of this study was to evaluate the effect of Cucurbitacin I (CuI) in a murine model of bleomycin-induced pulmonary fibrosis. MATERIAL Y METHODS Wistar rats were instilled intratracheally with a single dose of bleomycin (BLM)(3.75 U/kg; n=12) to induce lung fibrosis. CuI (20mg/kg/day; n=6) or CuI vehicle (control and IPF group) was administered intraperitoneally daily for 21 days. Animal evolution was controlled through CT/SPECT imaging. Tgf-β1, Col1A and Et-1 gene and protein expression was measured by real time PCR and western blott as fibrotic markers. RESULTS CT/SPECT quantification showed reduction of the fibrotic areas in the CuI treated group by reestablishing the air space in the lungs by inhibiting the IPF progression. Tgf-β1, Col1A and Et-1 gene expression was upregulated 2.3, 7.2 and 1.9 fold respectively in BLM relative to Control rats. This was counteracted with CuI treatment. These results were substantiated by protein over-expression of Tgf-β1 (62.30%), Col1A (45.80%) and Et-1 (44.44%) in BLM rat. Protein expression was inhibited in CuI treated group by 32.04%, 30.52% and 44.99% respectively. CONCLUSION CuI inhibits the rat model BLM induced pulmonary fibrosis.
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Papers by Javier Milara