The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform
Satish K. Srivastava - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A, Satish K. SrivastavaAbstract:Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-kB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be antiinflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, Ragweed Pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. Methods and Findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on Ragweed Pollen Extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE-induced apoptotic cell death as measured by annexin-v staining, increase in the activation o
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AR inhibition prevents RWE-induced expression of cytokine and chemokines in mice lungs.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:Total RNA was isolated from homogenized lungs (n = 5–7), one microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted for selected genes using specific forward and reverse primers. The levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (B). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear Extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p
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Aldose reductase deficiency in mice protects from Ragweed Pollen Extract (RWE)-induced allergic asthma
Respiratory Research, 2011Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:Background Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR^-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of Ragweed Pollen Extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat. Methods The wild type (WT) and AR^-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4^+CD25^+ T cells population. Results Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR^-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4^+CD25^+FoxP3^+) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat. Conclusion Our results using AR^-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.
Istvan Boldogh - One of the best experts on this subject based on the ideXlab platform.
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Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.
American journal of respiratory cell and molecular biology, 2016Co-Authors: Koa Hosoki, Leopoldo Aguilera-aguirre, Istvan Boldogh, Allan R. Brasier, Alexander Kurosky, Sanjiv SurAbstract:Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to Pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate Pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that Ragweed Pollen Extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induc...
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Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A, Satish K. SrivastavaAbstract:Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-kB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be antiinflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, Ragweed Pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. Methods and Findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on Ragweed Pollen Extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE-induced apoptotic cell death as measured by annexin-v staining, increase in the activation o
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Aldose Reductase Inhibition Prevents Metaplasia of Airway Epithelial Cells
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish KAbstract:Background: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Methodology/Findings: Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of Ragweed Pollen Extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE
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AR inhibition prevents RWE-induced expression of cytokine and chemokines in mice lungs.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:Total RNA was isolated from homogenized lungs (n = 5–7), one microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted for selected genes using specific forward and reverse primers. The levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (B). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
Umesh C. S. Yadav - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A, Satish K. SrivastavaAbstract:Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-kB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be antiinflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, Ragweed Pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. Methods and Findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on Ragweed Pollen Extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE-induced apoptotic cell death as measured by annexin-v staining, increase in the activation o
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Aldose Reductase Inhibition Prevents Metaplasia of Airway Epithelial Cells
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish KAbstract:Background: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Methodology/Findings: Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of Ragweed Pollen Extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE
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AR inhibition prevents RWE-induced expression of cytokine and chemokines in mice lungs.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:Total RNA was isolated from homogenized lungs (n = 5–7), one microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted for selected genes using specific forward and reverse primers. The levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (B). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear Extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p
Leopoldo Aguilera-aguirre - One of the best experts on this subject based on the ideXlab platform.
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Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.
American journal of respiratory cell and molecular biology, 2016Co-Authors: Koa Hosoki, Leopoldo Aguilera-aguirre, Istvan Boldogh, Allan R. Brasier, Alexander Kurosky, Sanjiv SurAbstract:Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to Pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate Pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that Ragweed Pollen Extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induc...
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Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A, Satish K. SrivastavaAbstract:Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-kB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be antiinflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, Ragweed Pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. Methods and Findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on Ragweed Pollen Extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE-induced apoptotic cell death as measured by annexin-v staining, increase in the activation o
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Aldose Reductase Inhibition Prevents Metaplasia of Airway Epithelial Cells
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish KAbstract:Background: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Methodology/Findings: Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of Ragweed Pollen Extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE
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AR inhibition prevents RWE-induced expression of cytokine and chemokines in mice lungs.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:Total RNA was isolated from homogenized lungs (n = 5–7), one microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted for selected genes using specific forward and reverse primers. The levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (B). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
Kota V. Ramana - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A, Satish K. SrivastavaAbstract:Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-kB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be antiinflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, Ragweed Pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. Methods and Findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on Ragweed Pollen Extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE-induced apoptotic cell death as measured by annexin-v staining, increase in the activation o
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Aldose Reductase Inhibition Prevents Metaplasia of Airway Epithelial Cells
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish KAbstract:Background: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Methodology/Findings: Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of Ragweed Pollen Extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE
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AR inhibition prevents RWE-induced expression of cytokine and chemokines in mice lungs.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:Total RNA was isolated from homogenized lungs (n = 5–7), one microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted for selected genes using specific forward and reverse primers. The levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Satish K. SrivastavaAbstract:The mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 µm. The sections were immunostained with p-STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB –based HRP conjugated antibodies counterstained with hematoxylin and eosin (B). Photomicrographs were acquired by fluorescence or light microscopes. A representative field for each group is shown (magnification: 200×). In (A) inset shows magnified view of the selected regions from representative photomicrographs (n = 4). RWE, Ragweed Pollen Extract; ARI, aldose reductase inhibitor.
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Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.
2013Co-Authors: Umesh C. S. Yadav, Leopoldo Aguilera-aguirre, Istvan Boldogh, Kota V. Ramana, Hamid A. Boulares, Satish K. SrivastavaAbstract:(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear Extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, Ragweed Pollen Extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p