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Effects Of Formoterol Stereoisomers On Airway Remodeling And Fibrinolytic System In A Murine Asthma Model
177
Modulation of ERK1/2 Levels by Enantiomers of Formoterol in Human Airway Smooth Muscle Cells R. J. Hasan1, A. L. Reno1, E. A. Duru1, J. L. Parks1, R. A. Panettieri, Jr, 2, W. J. Calhoun1, B. T. Ameredes1; 1University of Texas Medical Branch, Galveston, TX, 2University of Pennsylvania Medical Center, Philadelphia, PA. RATIONALE: Beta-2 adrenoreceptor agonists can modulate proliferation and release of cytokines by human airway smooth muscle cells (HASMC). The mechanism may be associated with MAPK signaling; however, the effects may be dependent on cell stimulation state and agonist enantiomer composition. Therefore, we assessed whether serum provision altered the ability of formoterol enantiomers to modulate ERK1/2 activation. METHODS: Confluent HASMC were serum-starved for 24hr, and then either continued without serum or serum added, and stimulated with IL-1beta (1ng/ml) and TNF-alpha (100ng/ml) for 30 min, followed by addition (10nM and 10 mM) of either (R,R)-formoterol, (S,S)-formoterol, or racemic (R,R-,S,S)-formoterol (20 nM and 20 mM). Phospho-p42/44 MAPK protein expression, as an index of ERK1/2 activation, was determined by Western Blot analysis of cell lysates. RESULTS: In the absence of serum, 10nM and 10 mM R,R-formoterol significantly decreased ERK1/2 activation by 50-60%, as compared to S,S-formoterol. In the presence of serum, S,S-formoterol at 10 nM increased ERK1/2 activation by 70% over R,R-formoterol, whereas with racemic formoterol, activation was intermediate in magnitude, in the absence and presence of serum. CONCLUSIONS: These results suggest decreased ERK1/2 activation by (R,R)-formoterol, as compared to (S,S)-formoterol, in the presence and absence of serum in HASMC, or conversely, increased activation of ERK1/2 by (S,S)-formoterol. The intermediate activation observed with racemic formoterol suggests competitive or opposing effects of the enantiomers within the racemate. We conclude that activation of ERK1/2 in HASMC can be differentially modulated by formoterol enantiomers, which may be dependent on the stimulation state of the cells.
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Statins May Improve Asthma O. Pagovich, E. Wang, M. Lee-Wong; Beth Israel Medical Center, New York, NY. RATIONALE: Lipid lowering statins are used for primary and secondary prevention of cardiovascular disease. Statins may also have anti-inflammatory and immunomodulating properties important in asthma therapy. METHODS: A retrospective chart review of asthma clinic patients was done. Patients were included if they had no change in medications and dosages except for the addition of statins during the period studied. Asthma severity, albuterol use and peak flows (PF) were recorded at one and two months prior to and after initiation of statin therapy. PF measurements and albuterol use were analyzed using paired t-tests and signed rank tests respectively. Wilcoxon rank tests were used to compare changes in albuterol use and PF measurements between atorvastatin and simvastatin regimens. RESULTS: Seventy patients met the inclusion criteria of which 54% were prescribed atorvastatin and 41% simvastatin. No significant changes in albuterol use (p 5 0.52) or PF measurements (p 5 0.63) were observed between 1 and 2 months pre-statin therapy. A significant increase in PF (p<0.0001) and decrease in albuterol use (p<0.0001) was observed one month post-statin. No significant differences were found in PF improvement (p50.24) or in albuterol use (p50.36) among the two different statin regimens. CONCLUSIONS: Lipid lowering statins are being investigated for use in rheumatoid arthritis, diabetes mellitus, multiple sclerosis, lung transplant, SLE, cancer and other disorders. In our study, the addition of statins was associated with reduction in albuterol use and improvement in PF measurements. Statins may have a potential role in asthma therapy that needs further investigation.
179
Mucosal Eicosanoid Biosynthesis in Aspirin-Exacerbated Respiratory Disease (AERD) P. S. Farooque1, D. Roberts2, C. J. Corrigan1, T. H. Lee1; 1Kings College London, London, UNITED KINGDOM, 2Guy’s Hospital, London, UNITED KINGDOM. RATIONALE: Patients with AERD can tolerate cyclooxygenase-2 (COX2) inhibitors whereas cyclooxygenase-1 (COX-1) inhibitors induce symptoms. Since prostaglandin E2 (PGE2) abrogates these symptoms, thought to be caused by elevated cysteinyl leukotriene (CysLT) production, we hypothesised that there is a deficiency of PGE2 production reflecting absent/reduced COX-2 activity in patients with AERD compared to aspirin-tolerant (ATOL) individuals. To address this we developed an ex vivo model of AERD. METHODS: Nasal polyps from AERD (n510) and ATOL patients (n514) were digested with collagenase and isolated cells pre-incubated with COX-1 (aspirin) or COX-2 inhibitors (NS-398) or control at optimised concentrations for 30 minutes prior to adding eicosanoid substrate (arachidonic acid, AA) or medium control for a further 30 minutes. Supernatant PGE2 and CysLT concentrations were measured by ELISA. COX-1 and COX-2 mRNA expression were determined by qRT-PCR. RESULTS: CysLT production was significantly elevated in AERD compared to ATOL patients at baseline and after futher addition of AA (p< 0.01). Pre-incubation with aspirin but not NS-398 significantly further elevated CysLT production (p<0.01) in the AERD but not the ATOL patients. PGE2 production was similar in both groups at baseline and similarly elevated after addition of AA. Pre-incubation with aspirin and NS-398 significantly inhibited PGE2 production (p<0.001) in both groups. qRT-PCR confirmed COX-1 mRNA and COX-2 mRNA expression in nasal polyps cells to an equivalent degree in both groups. CONCLUSIONS: Respiratory mucosal cells from AERD and ATOL patients produce equivalent amounts of PGE2 which reflects the activities of COX-1 and COX-2 in both patient groups.
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Effects Of Formoterol Stereoisomers On Airway Remodeling And Fibrinolytic System In A Murine Asthma Model Z. Ma, J. P. Tovar, K. Y. C. Kwong; Harbor-UCLA Medical Center, Torrance, CA. RATIONALE: The initiation and development of airway remodeling is dependent on airway inflammation partially and regulated by the fibrinolytic system. We previously demonstrated that (S,S)-formoterol has pro-inflammatory effects whereas (R,R)-formoterol has anti-inflammatory properties. We thus hypothesize that (S,S)-formoterol augments airway remodeling by affecting the fibrinolytic system. METHODS: The murine asthma model was induced in Balb/c mice with ovalbumin. Asthmatic mice were given daily inhalation of (R,R)- or (S,S)formoterol for 1 week. Goblet cells in lung tissues were verified by Periodic Acid-Schiff staining. Total collagen content in the lung was evaluated by measuring hydroxyproline levels. The level of protein or activity of fibrin, plamsminogen activator inhibitor-1 (PAI-1), and tissue type plasminogen activator (tPA) was measured by ELISA. RESULTS: Ovalbumin challenged mice showed goblet cell hyperplasia in the lungs compared with healthy controls and it was augmented by (S,S)formoterol but not (R,R)-formoterol. Fibrin and collagen levels in the lungs were 1.8-fold and 5.5-fold greater in asthmatic mice than in the control mice respectively (P<0.01). (S,S)-formoterol treatment caused further augmentation of the deposition of fibrin and collagen (P<0.01). The level of PAI-1 protein and activity was increased by 4.3-fold and 8.1-fold respectively, while the level of tPA activity was decreased by 75% in asthmatic mice compared to healthy mice (P<0.001). (S,S)-formoterol treatment further elevated PAI-1 protein and activity by 56% and 63% respectively (P<0.01), but diminished tPA activity by 43% (P<0.05) compared to mice without treatment. CONCLUSIONS:(S,S)-formoterol treatment exacerbates airway remodeling by decreasing the total fibrinolytic activity of lungs in the murine asthma model.
SATURDAY
Abstracts AB45
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
Modulation of ERK1/2 Levels by Enantiomers of Formoterol in Human Airway Smooth Muscle Cells R. J. Hasan1, A. L. Reno1, E. A. Duru1, J. L. Parks1, R. A. Panettieri, Jr, 2, W. J. Calhoun1, B. T. Ameredes1; 1University of Texas Medical Branch, Galveston, TX, 2University of Pennsylvania Medical Center, Philadelphia, PA. RATIONALE: Beta-2 adrenoreceptor agonists can modulate proliferation and release of cytokines by human airway smooth muscle cells (HASMC). The mechanism may be associated with MAPK signaling; however, the effects may be dependent on cell stimulation state and agonist enantiomer composition. Therefore, we assessed whether serum provision altered the ability of formoterol enantiomers to modulate ERK1/2 activation. METHODS: Confluent HASMC were serum-starved for 24hr, and then either continued without serum or serum added, and stimulated with IL-1beta (1ng/ml) and TNF-alpha (100ng/ml) for 30 min, followed by addition (10nM and 10 mM) of either (R,R)-formoterol, (S,S)-formoterol, or racemic (R,R-,S,S)-formoterol (20 nM and 20 mM). Phospho-p42/44 MAPK protein expression, as an index of ERK1/2 activation, was determined by Western Blot analysis of cell lysates. RESULTS: In the absence of serum, 10nM and 10 mM R,R-formoterol significantly decreased ERK1/2 activation by 50-60%, as compared to S,S-formoterol. In the presence of serum, S,S-formoterol at 10 nM increased ERK1/2 activation by 70% over R,R-formoterol, whereas with racemic formoterol, activation was intermediate in magnitude, in the absence and presence of serum. CONCLUSIONS: These results suggest decreased ERK1/2 activation by (R,R)-formoterol, as compared to (S,S)-formoterol, in the presence and absence of serum in HASMC, or conversely, increased activation of ERK1/2 by (S,S)-formoterol. The intermediate activation observed with racemic formoterol suggests competitive or opposing effects of the enantiomers within the racemate. We conclude that activation of ERK1/2 in HASMC can be differentially modulated by formoterol enantiomers, which may be dependent on the stimulation state of the cells.
178
Statins May Improve Asthma O. Pagovich, E. Wang, M. Lee-Wong; Beth Israel Medical Center, New York, NY. RATIONALE: Lipid lowering statins are used for primary and secondary prevention of cardiovascular disease. Statins may also have anti-inflammatory and immunomodulating properties important in asthma therapy. METHODS: A retrospective chart review of asthma clinic patients was done. Patients were included if they had no change in medications and dosages except for the addition of statins during the period studied. Asthma severity, albuterol use and peak flows (PF) were recorded at one and two months prior to and after initiation of statin therapy. PF measurements and albuterol use were analyzed using paired t-tests and signed rank tests respectively. Wilcoxon rank tests were used to compare changes in albuterol use and PF measurements between atorvastatin and simvastatin regimens. RESULTS: Seventy patients met the inclusion criteria of which 54% were prescribed atorvastatin and 41% simvastatin. No significant changes in albuterol use (p 5 0.52) or PF measurements (p 5 0.63) were observed between 1 and 2 months pre-statin therapy. A significant increase in PF (p<0.0001) and decrease in albuterol use (p<0.0001) was observed one month post-statin. No significant differences were found in PF improvement (p50.24) or in albuterol use (p50.36) among the two different statin regimens. CONCLUSIONS: Lipid lowering statins are being investigated for use in rheumatoid arthritis, diabetes mellitus, multiple sclerosis, lung transplant, SLE, cancer and other disorders. In our study, the addition of statins was associated with reduction in albuterol use and improvement in PF measurements. Statins may have a potential role in asthma therapy that needs further investigation.
179
Mucosal Eicosanoid Biosynthesis in Aspirin-Exacerbated Respiratory Disease (AERD) P. S. Farooque1, D. Roberts2, C. J. Corrigan1, T. H. Lee1; 1Kings College London, London, UNITED KINGDOM, 2Guy’s Hospital, London, UNITED KINGDOM. RATIONALE: Patients with AERD can tolerate cyclooxygenase-2 (COX2) inhibitors whereas cyclooxygenase-1 (COX-1) inhibitors induce symptoms. Since prostaglandin E2 (PGE2) abrogates these symptoms, thought to be caused by elevated cysteinyl leukotriene (CysLT) production, we hypothesised that there is a deficiency of PGE2 production reflecting absent/reduced COX-2 activity in patients with AERD compared to aspirin-tolerant (ATOL) individuals. To address this we developed an ex vivo model of AERD. METHODS: Nasal polyps from AERD (n510) and ATOL patients (n514) were digested with collagenase and isolated cells pre-incubated with COX-1 (aspirin) or COX-2 inhibitors (NS-398) or control at optimised concentrations for 30 minutes prior to adding eicosanoid substrate (arachidonic acid, AA) or medium control for a further 30 minutes. Supernatant PGE2 and CysLT concentrations were measured by ELISA. COX-1 and COX-2 mRNA expression were determined by qRT-PCR. RESULTS: CysLT production was significantly elevated in AERD compared to ATOL patients at baseline and after futher addition of AA (p< 0.01). Pre-incubation with aspirin but not NS-398 significantly further elevated CysLT production (p<0.01) in the AERD but not the ATOL patients. PGE2 production was similar in both groups at baseline and similarly elevated after addition of AA. Pre-incubation with aspirin and NS-398 significantly inhibited PGE2 production (p<0.001) in both groups. qRT-PCR confirmed COX-1 mRNA and COX-2 mRNA expression in nasal polyps cells to an equivalent degree in both groups. CONCLUSIONS: Respiratory mucosal cells from AERD and ATOL patients produce equivalent amounts of PGE2 which reflects the activities of COX-1 and COX-2 in both patient groups.
180
Effects Of Formoterol Stereoisomers On Airway Remodeling And Fibrinolytic System In A Murine Asthma Model Z. Ma, J. P. Tovar, K. Y. C. Kwong; Harbor-UCLA Medical Center, Torrance, CA. RATIONALE: The initiation and development of airway remodeling is dependent on airway inflammation partially and regulated by the fibrinolytic system. We previously demonstrated that (S,S)-formoterol has pro-inflammatory effects whereas (R,R)-formoterol has anti-inflammatory properties. We thus hypothesize that (S,S)-formoterol augments airway remodeling by affecting the fibrinolytic system. METHODS: The murine asthma model was induced in Balb/c mice with ovalbumin. Asthmatic mice were given daily inhalation of (R,R)- or (S,S)formoterol for 1 week. Goblet cells in lung tissues were verified by Periodic Acid-Schiff staining. Total collagen content in the lung was evaluated by measuring hydroxyproline levels. The level of protein or activity of fibrin, plamsminogen activator inhibitor-1 (PAI-1), and tissue type plasminogen activator (tPA) was measured by ELISA. RESULTS: Ovalbumin challenged mice showed goblet cell hyperplasia in the lungs compared with healthy controls and it was augmented by (S,S)formoterol but not (R,R)-formoterol. Fibrin and collagen levels in the lungs were 1.8-fold and 5.5-fold greater in asthmatic mice than in the control mice respectively (P<0.01). (S,S)-formoterol treatment caused further augmentation of the deposition of fibrin and collagen (P<0.01). The level of PAI-1 protein and activity was increased by 4.3-fold and 8.1-fold respectively, while the level of tPA activity was decreased by 75% in asthmatic mice compared to healthy mice (P<0.001). (S,S)-formoterol treatment further elevated PAI-1 protein and activity by 56% and 63% respectively (P<0.01), but diminished tPA activity by 43% (P<0.05) compared to mice without treatment. CONCLUSIONS:(S,S)-formoterol treatment exacerbates airway remodeling by decreasing the total fibrinolytic activity of lungs in the murine asthma model.
SATURDAY
Abstracts AB45
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2