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O57. Exhaled NO—From basic discovery to clinical use
Oral abstracts / Nitric Oxide 19 (2008) S20–S42 Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused
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pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and inhibited by chloral hydrate (both isoforms) and daidzin (ALDH2). GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 lM in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. Since ALDH1 and ALDH2 enhanced activation of sGC by inorganic nitrite in a chloral hydrate-sensitive manner, GTN-derived nitrite may be converted into NO radical at the catalytic site of the enzymes. doi:10.1016/j.niox.2008.06.057
Inhaled and Exhaled NO O57. Exhaled NO—From basic discovery to clinical use Eddie Weitzberg Karolinska Institutet The presence of NO in exhaled air of humans was first described in 1991 and 2 years later it was shown that increased levels of NO were indicative of lower airway inflammation. Since then extensive research has led to the use of exhaled NO in experimental research and clinical practise. It is a simple, rapid and non-invasive test included in several clinical guidelines for diagnosis and monitoring of asthma, Since the nasal cavity and the paranasal sinuses also are main contributors to exhaled NO, measurements of NO from these sites is of importance. I will give an overview on how this field has developed from discovery to clinical use and portray different aspects on how exhaled NO can be utilized in experimental research as well as in clinical practise. doi:10.1016/j.niox.2008.06.058
O58. Exhaled NO and inflammatory disorders affecting lung alveoli and the distal airways Anh Tuan Dinh-Xuan Paris Descartes University & Cochin Hospital Alveolar inflammation associated with connective tissue disease, such as systemic sclerosis (SSc), increases NO production in the distal part of the lung. Modelling lung gas exchange and partitioning exhaled NO allow non invasive assessment of inflammation in lung alveoli and distal airways. Interstitial lung disease (ILD) resulting from chronic insult to lung tissue has become the main cause of death in SSc. Although the underlying mechanisms of SSc-associated ILD remain unclear, it is known that immune activation leads to alveolar inflammation and the release of several pro-inflammatory cytokines, which in turn trigger activation of inducible NO synthase and production of high amounts of NO. It is speculated that overproduction of NO in the lung eventually leads to ILD in SSc patients. Previous observations have reported increased exhaled NO (from airways and alveoli) in patients with SSc with (or without) lung disease. Using the two-compartment model method and partitioning exhaled NO into alveolar concentration (CANO) and conducting airway flux, we have found that CANO is significantly increased in SSc patients as compared with healthy controls. We have also demonstrated that high levels of CANO were related to the severity of ILD in SSc patients. To determine the clinical usefulness of partitioned measurement of exhaled NO in ILD, we have determined the threshold values of CANO which could help to identify patients SSc with ILD. We have found that ILD could be ruled in (positive predictive value >95%) when CANO 10.5 ppb, and ruled out CANO values 3.8 ppb (negative predictive value >95%). Finally, to identify the underlying pathways that govern the relationship between increased CANO and ILD, we have concomitantly measured CANO levels and lung fibroblasts proliferation induced by the serum from SSc patients. The serum ability to induce lung fibroblast proliferation and myofibroblast transition was increased in SSc patients with high levels of CANO (>5 ppb) as compared to SSc patients with low levels of CANO (5 ppb) and healthy controls. Our findings suggest a possible link between alveolar inflammation, reflected by the levels of alveolar NO output, and lung proliferation, assessed by fibroblast proliferation and myofibroblast transition, in SSc patients with ILD, a fearful condition that considerably worsens the prognosis of the disease, still as yet has no cure. doi:10.1016/j.niox.2008.06.059
O59. Impaired NO-cGMP signaling in neonatal pulmonary hypertension: Novel mechanisms and therapies Steven H. Abman Department of Pediatrics, University of Colorado School of Medicine Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by failure of the lung circulation to achieve or sustain the normal drop in pulmonary vascular resistance (PVR) at birth. Past laboratory studies identified the important role of nitric oxide (NO)-cGMP signaling in the regulation of the perinatal lung circulation, leading to the development and application of inhaled NO therapy for PPHN. Although iNO therapy has improved the clinical course and outcomes of many infants, pulmonary hypertension can be refractory to inhaled NO, suggesting the need for additional approaches to severe PPHN. To develop novel therapeutic strategies for PPHN, ongoing studies continue to explore basic mechanisms underlying the pathobiology of PPHN in experimental models, including strategies to enhance NO-cGMP signaling. Recent studies have demonstrated that impaired vascular endothelial growth factor (VEGF) signaling down-regulates eNOS expression and may contribute to the pathogenesis of PPHN. Other studies have shown that enhanced NO-cGMP activity through the use of cGMP-specific phosphodiesterase inhibitors (sildenafil), soluble guanylate cyclase activators (BAY 41-2272 and BAY-58-2667), superoxide scavengers (superoxide dismutase), and rho kinase inhibitors (fasudil) can lead to potent and sustained pulmonary vasodilation and improved vascular growth in experimental PPHN. Overall, these laboratory studies suggest novel pharmacologic strategies for the treatment of refractory PPHN, and highlight the need for further clinical studies of augmentation of NO-cGMP signaling in the treatment of sick neonates with PPHN. doi:10.1016/j.niox.2008.06.060
O60. Exhaled breath condensate nitrite and plasma nitrite. Pre- and postoperative comparative study in patients submitted to cardiopulmonary bypass Viviane dos Santos Augusto, Graziela Saraiva Reis, Maria Eliza Jordani de Souza, Alfredo José Rodrigues, Paulo Roberto Barbosa Evora Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo Context and aims: Exhaled NO has been considered as a potential pulmonary injury biomarker after on-pump cardiac surgery and the use of the exhaled breath condensate (EBC) as a non-invasive method to investigate pulmonary injuries has gained progressive interest. By means of this prospective and nonrandomized study, the author aimed to verify the possible association between the levels of plasma and condensed pulmonary exhale nitrite (NO2 ) in preoperatory and late post-operatory (24 h) periods of on-pump cardiac surgery. Methods: Twenty-eight adult patients of both genders and between 26 and 71 years of age were selected and subdivided into two groups: (1) control (non-surgery) and (2) surgery (valve surgery and myocardium revascularization). EBC and blood samples of each and every patient were frozen and stored at 70 °C. Plasma and EBC nitrite levels were performed by the chemiluminescense method (Nitric Oxide Analyzer, 280i, SIEVERS-NOA-Sievers). Data are presented as means and standard deviation, and for statistical analysis the non-parametric tests of Mann–Whitney and Wilcoxon were adopted. Results: The main results were: (a) the levels of EBC NO2 collected with dry ice on the pre-operatory surgery group were higher than on control group collected with the same
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pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and inhibited by chloral hydrate (both isoforms) and daidzin (ALDH2). GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 lM in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. Since ALDH1 and ALDH2 enhanced activation of sGC by inorganic nitrite in a chloral hydrate-sensitive manner, GTN-derived nitrite may be converted into NO radical at the catalytic site of the enzymes. doi:10.1016/j.niox.2008.06.057
Inhaled and Exhaled NO O57. Exhaled NO—From basic discovery to clinical use Eddie Weitzberg Karolinska Institutet The presence of NO in exhaled air of humans was first described in 1991 and 2 years later it was shown that increased levels of NO were indicative of lower airway inflammation. Since then extensive research has led to the use of exhaled NO in experimental research and clinical practise. It is a simple, rapid and non-invasive test included in several clinical guidelines for diagnosis and monitoring of asthma, Since the nasal cavity and the paranasal sinuses also are main contributors to exhaled NO, measurements of NO from these sites is of importance. I will give an overview on how this field has developed from discovery to clinical use and portray different aspects on how exhaled NO can be utilized in experimental research as well as in clinical practise. doi:10.1016/j.niox.2008.06.058
O58. Exhaled NO and inflammatory disorders affecting lung alveoli and the distal airways Anh Tuan Dinh-Xuan Paris Descartes University & Cochin Hospital Alveolar inflammation associated with connective tissue disease, such as systemic sclerosis (SSc), increases NO production in the distal part of the lung. Modelling lung gas exchange and partitioning exhaled NO allow non invasive assessment of inflammation in lung alveoli and distal airways. Interstitial lung disease (ILD) resulting from chronic insult to lung tissue has become the main cause of death in SSc. Although the underlying mechanisms of SSc-associated ILD remain unclear, it is known that immune activation leads to alveolar inflammation and the release of several pro-inflammatory cytokines, which in turn trigger activation of inducible NO synthase and production of high amounts of NO. It is speculated that overproduction of NO in the lung eventually leads to ILD in SSc patients. Previous observations have reported increased exhaled NO (from airways and alveoli) in patients with SSc with (or without) lung disease. Using the two-compartment model method and partitioning exhaled NO into alveolar concentration (CANO) and conducting airway flux, we have found that CANO is significantly increased in SSc patients as compared with healthy controls. We have also demonstrated that high levels of CANO were related to the severity of ILD in SSc patients. To determine the clinical usefulness of partitioned measurement of exhaled NO in ILD, we have determined the threshold values of CANO which could help to identify patients SSc with ILD. We have found that ILD could be ruled in (positive predictive value >95%) when CANO 10.5 ppb, and ruled out CANO values 3.8 ppb (negative predictive value >95%). Finally, to identify the underlying pathways that govern the relationship between increased CANO and ILD, we have concomitantly measured CANO levels and lung fibroblasts proliferation induced by the serum from SSc patients. The serum ability to induce lung fibroblast proliferation and myofibroblast transition was increased in SSc patients with high levels of CANO (>5 ppb) as compared to SSc patients with low levels of CANO (5 ppb) and healthy controls. Our findings suggest a possible link between alveolar inflammation, reflected by the levels of alveolar NO output, and lung proliferation, assessed by fibroblast proliferation and myofibroblast transition, in SSc patients with ILD, a fearful condition that considerably worsens the prognosis of the disease, still as yet has no cure. doi:10.1016/j.niox.2008.06.059
O59. Impaired NO-cGMP signaling in neonatal pulmonary hypertension: Novel mechanisms and therapies Steven H. Abman Department of Pediatrics, University of Colorado School of Medicine Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by failure of the lung circulation to achieve or sustain the normal drop in pulmonary vascular resistance (PVR) at birth. Past laboratory studies identified the important role of nitric oxide (NO)-cGMP signaling in the regulation of the perinatal lung circulation, leading to the development and application of inhaled NO therapy for PPHN. Although iNO therapy has improved the clinical course and outcomes of many infants, pulmonary hypertension can be refractory to inhaled NO, suggesting the need for additional approaches to severe PPHN. To develop novel therapeutic strategies for PPHN, ongoing studies continue to explore basic mechanisms underlying the pathobiology of PPHN in experimental models, including strategies to enhance NO-cGMP signaling. Recent studies have demonstrated that impaired vascular endothelial growth factor (VEGF) signaling down-regulates eNOS expression and may contribute to the pathogenesis of PPHN. Other studies have shown that enhanced NO-cGMP activity through the use of cGMP-specific phosphodiesterase inhibitors (sildenafil), soluble guanylate cyclase activators (BAY 41-2272 and BAY-58-2667), superoxide scavengers (superoxide dismutase), and rho kinase inhibitors (fasudil) can lead to potent and sustained pulmonary vasodilation and improved vascular growth in experimental PPHN. Overall, these laboratory studies suggest novel pharmacologic strategies for the treatment of refractory PPHN, and highlight the need for further clinical studies of augmentation of NO-cGMP signaling in the treatment of sick neonates with PPHN. doi:10.1016/j.niox.2008.06.060
O60. Exhaled breath condensate nitrite and plasma nitrite. Pre- and postoperative comparative study in patients submitted to cardiopulmonary bypass Viviane dos Santos Augusto, Graziela Saraiva Reis, Maria Eliza Jordani de Souza, Alfredo José Rodrigues, Paulo Roberto Barbosa Evora Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo Context and aims: Exhaled NO has been considered as a potential pulmonary injury biomarker after on-pump cardiac surgery and the use of the exhaled breath condensate (EBC) as a non-invasive method to investigate pulmonary injuries has gained progressive interest. By means of this prospective and nonrandomized study, the author aimed to verify the possible association between the levels of plasma and condensed pulmonary exhale nitrite (NO2 ) in preoperatory and late post-operatory (24 h) periods of on-pump cardiac surgery. Methods: Twenty-eight adult patients of both genders and between 26 and 71 years of age were selected and subdivided into two groups: (1) control (non-surgery) and (2) surgery (valve surgery and myocardium revascularization). EBC and blood samples of each and every patient were frozen and stored at 70 °C. Plasma and EBC nitrite levels were performed by the chemiluminescense method (Nitric Oxide Analyzer, 280i, SIEVERS-NOA-Sievers). Data are presented as means and standard deviation, and for statistical analysis the non-parametric tests of Mann–Whitney and Wilcoxon were adopted. Results: The main results were: (a) the levels of EBC NO2 collected with dry ice on the pre-operatory surgery group were higher than on control group collected with the same