Inhaled nitric oxide alleviates hyperoxia suppressed phosphatidylcholine synthesis in endotoxin-induced injury in mature rat lungs

BACKGROUND: We investigated efficacy of inhaled nitric oxide (NO) in modulation of metabolism of phosphatidylcholine (PC) of pulmonary surfactant and in anti-inflammatory mechanism of mature lungs with inflammatory injury. METHODS: Healthy adult rats were divided into a group of lung inflammation induced by i.v. lipopolysaccharides (LPS) or a normal control (C) for 24 h, and then exposed to: room air (Air), 95% oxygen (O), NO (20 parts per million, NO), both O and NO (ONO) as subgroups, whereas [(3)H]-choline was injected i.v. for incorporation into PC of the lungs which were processed subsequently at 10 min, 4, 8, 12 and 24 h, respectively, for measurement of PC synthesis and proinflammatory cytokine production. RESULTS: LPS-NO subgroup had the lowest level of labeled PC in total phospholipids and disaturated PC in bronchoalveolar lavage fluid and lung tissue (decreased by 46–59%), along with the lowest activity of cytidine triphosphate: phosphocholine cytidylyltransferase (-14–18%) in the lungs, compared to all other subgroups at 4 h (p < 0.01), but not at 8 and 12 h. After 24-h, all LPS-subgroups had lower labeled PC than the corresponding C-subgroups (p < 0.05). LPS-ONO had higher labeled PC in total phospholipids and disaturated PC, activity of cytidylyltransferase, and lower activity of nuclear transcription factor-κB and expression of proinflammatory cytokine mRNA, than that in the LPS-O subgroup (p < 0.05). CONCLUSION: In LPS-induced lung inflammation in association with hyperoxia, depressed PC synthesis and enhanced proinflammatory cytokine production may be alleviated by iNO. NO alone only transiently suppressed the PC synthesis as a result of lower activity of cytidylyltransferase

Determinantes estructurales de la actividad biológica de Fas ligando soluble en la lesión pulmonar agudo

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Medicina. Fecha de lectura: 29 de Septiembre 200

Role of iNOS in septic pulmonary microvascular endothelial cell activation

Abstract Background. Neutrophils and nitric oxide (NO) derived from inducible NO synthase (iNOS) contributes importantly to the pathophysiology of acute lung injury (ALI) and pulmonary microvascular endothelial cell (PMVEC) injury. However, the mechanism of neutrophil and neutrophil iNOS dependent PMVEC injury has not been addressed. In our studies, we assessed PMVEC activation under septic conditions, and defined the role of PMVEC vs. bone-marrow polymorphonuclear leukocytes (PMN) iNOS in this septic PMVEC activation. Methods and Results. We isolated PMVEC from iNOS+/+ and iNOS-/- mice lungs magnetically by microbeads attached to anti-PECAM antibodies, sorted by flow cytometry (FACS) by DiI-acetylated low density lipoprotein (Dil-Ac-LDL) uptake by cells. Bone-marrow PMNs were isolated from femurs and tibia of iNOS+/+ and iNOS-/- mice followed by percoll gradient isolation. Cultured PMVEC monolayers showed that basal E-selectin expression was significantly lower in iNOS-/- vs iNOS+/+ PMVEC (MFI 16±2 vs 59±7, p\u3c0.05). After lipopolysaccharide (LPS) stimulation, E-selectin expression in iNOS+/+ PMVEC increased at 1 hr (MFI 146±15, p\u3c0.01), peaked at 2 hrs (MFI 284±38, p\u3c0.01), and then gradually declined over 12 hrs. iNOS-/- PMVEC had a similar response to iNOS+/+ PMVEC in the timing and magnitude of E-selectin upregulation, with a similar peak expression (MFI 257±29, p=NS vs iNOS+/+). PMVEC responded to cytomix similarly to LPS, with PMVEC E-selectin upregulation in a time dependent and dose dependent manner which was similar in iNOS+/+ and iNOS-/- PMVEC. As compared to E-selectin, basal plasminogen activation inhibitor (PAI)-1 expression was similar in iNOS+/+ and iNOS-/- PMVEC. LPS-induced upregulation of PAI-1 in iNOS+/+ PMVEC peaked at 4 hrs (MFI 3.4±0.1 vs 1.7±0.1 for control medium, p\u3c0.01), and was sustained until 12 hrs and this pattern of LPS-stimulated PAI-1 expression was similar in iNOS-/- PMVEC. In contrast, cytomix induced PAI-1 in iNOS+/+ PMVEC peaked at 2 hrs and was sustained until 8 hrs, and was similar in iNOS-/- PMVEC. The presence of PMN with PMVEC synergistically enhanced LPS induced PMVEC E-selectin expression in a dose dependent manner. However, this LPS-induced PMN-dependent PMVEC E-selectin induction was independent of PMN iNOS. The presence of either iNOS+/+ or iNOS-/- PMN with PMVEC did not upregulate PMVEC E-selectin post cytomix stimulation. Moreover, iNOS+/+ or iNOS-/- PMN did not affect PMVEC PAI-1 protein expression or mRNA expression after either LPS or cytomix treatment. Conclusions. PMVEC are activated under different septic conditions (both LPS and cytomix) as reflected by increased E-selectin and PAI-1 expression. However, PMVEC iNOS did not affect LPS or cytomix induced PMVEC activation. PMN enhanced septic PMVEC E-selectin expression following LPS treatment, and this endothelial activation was independent of PMN iNOS. These data suggest that septic PMVEC activation in isolation or in the presence of PMN, is independent of both PMVEC and PMN iNOS

The preemptive use of inhaled nitric oxide during cardiopulmonary bypass in an experimental pig model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Expression of haem oxygenase-1 and inducible nitric oxide synthase in horses with lower airway inflammation

The heat shock protein haem oxygenase-1 (HO-1) and the enzyme inducible nitric oxide synthase (iNOS) are both induced during conditions of oxidative stress and by pro-inflammatory mediators. Both HO-1 and iNOS may therefore be induced by a range of disease states that result in tissue inflammation. Both have been demonstrated to have potentially beneficial anti-inflammatory effects. Both HO-1 and iNOS and their respective gaseous products carbon monoxide and nitric oxide have being the subject of investigation as potential therapeutic targets for human respiratory disease, most notably asthma. The pathogenesis of inflammatory diseases of the equine lower respiratory tract has many similarities with human asthma and both HO-1 and iNOS have been demonstrated in fixed equine pulmonary tissue. The aim of the study was to investigate the expression of HO-1 and iNOS in the equine respiratory tract. The primary objective was to investigate whether HO-1 and iNOS were expressed within leukocytes from equine BALF and to semi-quantify expression in different cell types. If it proved possible to characterise and quantify expression, additional aims were to investigate associations between HO-1 expression, iNOS expression, clinical signs of respiratory disease and markers of oxidative stress in exhaled breath condensate. We were successful in demonstrating the expression of both HO-1 and iNOS in leukocytes from equine BALF. Expression was most intense in macrophages and neutrophils which was consistent with previous studies performed in other species. We did not identify an association between the degree of HO-1 or iNOS staining in leukocytes from BALF and the severity of clinical respiratory disease. We also failed to identify an association between expression of HO-1 and iNOS in leukocytes from BALF and markers of oxidative stress in exhaled breath condensate. Further investigations ought to be performed into the expression and activity of both HO-1 and iNOS in the equine respiratory tract

Pulmonary Protection Strategies in Cardiac Surgery: Are We Making Any Progress?

Pulmonary dysfunction is a common complication of cardiac surgery. The mechanisms involved in the development of pulmonary dysfunction are multifactorial and can be related to the activation of inflammatory and oxidative stress pathways. Clinical manifestation varies from mild atelectasis to severe respiratory failure. Managing pulmonary dysfunction postcardiac surgery is a multistep process that starts before surgery and continues during both the operative and postoperative phases. Different pulmonary protection strategies have evolved over the years; however, the wide acceptance and clinical application of such techniques remain hindered by the poor level of evidence or the sample size of the studies. A better understanding of available modalities and/or combinations can result in the development of customised strategies for the different cohorts of patients with the potential to hence maximise patients and institutes benefits

Surfactant phospholipid metabolism and respiratory failure in lung transplantation

Primary graft dysfunction (PGD) after lung transplantation is a multi-factorial syndrome of postoperative lung injury that is similar to the acute respiratory distress syndrome. PGD is a major risk factor for chronic lung allograft dysfunction and perioperative death. Pulmonary surfactant dysfunction is frequently seen in PGD and contributes to respiratory failure. Surfactant is primarily comprised of lipids, of which phosphatidylcholine (PC) species predominate. Dipalmitoylphosphatidylcholine (DPPC) is the most surface-active species that normally represents over half of all airway PC content. Combining electrospray ionisation tandem mass spectrometry with deuterium labelling of Kennedy’s PC synthetic pathway can assess surfactant phospholipid flux, with a molecular specificity that differentiates between lipids with unique functions. This technique was used to quantify perioperative phospholipid metabolic disruption in lung transplantation. The same technique was used in an ex vivo human lung model to isolate the pulmonary contribution to systemic phospholipid metabolism, and evaluate Kennedy pathway integrity during ex vivo lung perfusion (EVLP). Airway DPPC content was severely reduced after transplantation, and was associated with impaired oxygenation. Variations in Kennedy pathway activity were associated with impaired graft function for months after transplantation. Perioperative flux of phospholipids, like DPPC and polyunsaturated PC species, were strong predictors of poor postoperative lung function. Deuterium labelled ex vivo experiments confirmed continued Kennedy pathway dependent surfactant phospholipid synthesis during EVLP, and could represent a protective mechanism against PGD in clinical EVLP. Ex vivo lungs secreted a different phospholipid profile into the perfusate that is enriched with polyunsaturated PC when compared to the airways, providing compelling evidence of the previously hypothesized basolateral phospholipid secretion by the alveolar epithelium. These polyunsaturated PC species are precursors of numerous inflammatory mediators, and could contribute to PGD. Continued investigation of phospholipid flux through the Kennedy pathway could identify novel biomarkers and therapeutic targets in lung transplantation.Open Acces

Mass Spectrometry-Based High Throughput Approach for Identification of Molecular Modification of Oxidative Process in Respiratory Diseases

Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) have been implicated in generating reactive species and promoting oxidative modifications in numerous diseases. The detection and identification of potential pathways has been proven extremely challenge due to evanescence nature of these reactive species. An alternative approach to study the involvement of oxidative modification is to detect and quantify the stable molecular fingerprints, like oxidized tyrosine species, in biological matrices. Previously reported analytical methods for quantifying oxidized amino acids have typically been limited by: low sensitivity, specificity and the failure of detection potential oxidation products generated during sample handling. Using GC/MS in combination with stable isotope dilution, a sensitive and self-quality control assay was developed. Using this isotope-dilution GC/MS and multiple allergen challenge models, we demonstrate that EPO is the major pathway for generating protein-bound 3-bromotyrosine in vivo. However, the need for chemical derivatization of samples increases the possibility that artifactual generation of oxidation products which may interfere with the detection of native levels. Requirement of unique derivatization reagent for each oxidized tyrosine results sample analysis both cumbersome and time/labor intensive. Therefore, we develop a liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) assay that simultaneously detects and quantifies multiple structurally informative protein oxidation products along with their precursor amino acids. Use of four HPLC systems in multiplex array with column switching permits on-line analyses of only relevant portions of chromatographic profiles and up to four-fold increased throughput efficiency of mass detector usage. Using this high-throughput multiplexed array-based LC-MS/MS method, we found significant correlations among individual oxidized tyrosine in sera from healthy volunteers. These correlations provide chemica