PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

Are sputum eosinophils associated with a different phenotype in COPD patients? A retrospective study.

Sputum eosinophilia in Chronic Obstructive Pulmonary Disease (COPD) patients seems to be associated with a better response to inhaled corticosteroids (ICS). To verify if this feature could identify a specific subpopulation of COPD patients, we retrospectively compared functional and inflammatory parameters of 110 COPD patients according to the presence of sputum eosinophilia (>2%). Patient with eosinophilia were characterized by lower dyspnea score, lower functional impairment and lower ICS use, suggesting that airway eosinophilia may be associated to a lower COPD severity and some functional "asthma-like" characteristics, therefore explaining the better response to ICS in this subgroup of patients.

Upper and lower airway inflammation in severe asthmatics: a guide for a precision biologic treatment.

BACKGROUND AND AIMS: Severe asthma may require the prescription of one of the biologic drugs currently available, using surrogate markers of airway inflammation (serum IgE levels and allergic sensitization for anti-IgE, or blood eosinophils for anti-IL5/IL5R). Our objective: to assess upper and lower airway inflammation in severe asthmatics divided according to the eligibility criteria for one of the target biologic treatments. METHODS: We selected 91 severe asthmatics, uncontrolled despite high-dose ICS-LABA, and followed for >6 months with optimization of asthma treatment. Patients underwent clinical, functional and biological assessment, including induced sputum and nasal cytology. They were then clustered according to the eligibility criteria for omalizumab or mepolizumab/benralizumab. RESULTS: Four clusters were selected: A (eligible for omalizumab, n = 23), AB (both omalizumab and mepolizumab, n = 26), B (mepolizumab, n = 22) and C (non-eligible for both omalizumab and mepolizumab, n = 20). There was no difference among clusters for asthma control (Asthma Control Test and Asthma Control Questionnaire 7), pre-bronchodilator forced expiratory volume in 1 s, serum IgE and fractional exhaled nitric oxide levels. Sputum eosinophils were numerically higher in clusters AB and B, in agreement with the higher levels of blood eosinophils. Allergic rhinitis was more frequent in clusters A and AB, while chronic rhinosinusitis with nasal polyps prevalence increased progressively from A to C. Eosinophils in nasal cytology were higher in clusters AB, B and C. CONCLUSION: Eosinophilic upper and lower airway inflammation is present in the large majority of severe asthmatics, independently from the prescription criteria for the currently available biologics, and might suggest the use of anti-IL5/IL5R or anti IL4/13 also in patients without blood eosinophilia.The reviews of this paper are available via the supplemental material section.

Distinct profile of inflammatory and remodelling biomarkers in sputum of severe asthmatic patients with or without persistent airway obstruction.

BACKGROUND: Both inflammatory and remodelling processes are associated with irreversible airway obstruction observed in severe asthma. Our aim was to characterize a group of severe asthmatic patients with or without persistent airway obstruction in relation to specific sputum inflammatory and remodelling biomarkers. METHODS: Forty-five patients under regular high-dose inhaled corticosteroid/ß-2agonist treatment were studied, after a follow-up period of at least 2 years, with a minimum of 4 visits. Periostin, TGF-ß, RANTES, IL-8, GM-CSF, FGF-2, and cell counts were measured in induced sputum. Serum periostin was also measured. RESULTS: Sputum induction was successfully performed in all but 5 patients. There were no significant differences in demographic and clinical data between patients with non-persistent obstruction (NO: FEV1/VC>88%pred.) and those with persistent obstruction (O: a not completely reversible obstruction with FEV1/VC<88%pred. at each visit before the study visit). Patients with persistent obstruction had significantly higher sputum periostin and TGF-ß concentrations than NO patients and a trend of higher serum periostin levels. GM-CSF and FGF-2 were significantly increased in NO compared to O patients. No differences between groups were found for RANTES, IL-8 and differential cell counts. Sputum periostin inversely correlated with functional parameters (prebronch. FEV1: rho = -0.36, p < 0.05; postbronch. FEV1: rho = -0.33, p = 0.05). Patients with high sputum periostin concentration (>103.3 pg/ml: median value) showed an absolute number of sputum eosinophils significantly higher than patients with low sputum periostin; this behavior was unobserved when serum periostin was considered. CONCLUSIONS: Only periostin and TGF-ß identified a subgroup of severe asthmatic patients with persistent airway obstruction. Sputum periostin was also inversely associated with FEV1 and proved to be a more sensitive biomarker than serum periostin to identify severe asthmatics with higher sputum eosinophilia.

Comorbidities are associated with different features of severe asthma.

BACKGROUND: According to ATS/ERS document on severe asthma (SA), the management of these patients requires the identification and proper treatment of comorbidities, which can influence the control of asthma. METHODS: The aim of this study was to assess the independent effect of different comorbidities on clinical, functional and biologic features of SA. Seventy-two patients with SA according to GINA guidelines were examined. We collected demographic data, smoking habit, asthma history, and assessment of comorbidities. Pulmonary function, inflammatory biomarkers, upper airway disease evaluation, asthma control and quality of life were carefully assessed. RESULTS: The mean age of patients was 59.1 years (65.3% female, 5.6% current smokers). Comorbidities with higher prevalence were: chronic rhinosinusitis with or without nasal polyps (CRSwNP or CRSsNP), obesity and gastro-esophageal reflux (GERD), with some overlapping among them. In an univariate analysis comparing patients with single comorbidities with the other ones, asthmatics with CRSwNP had lower lung function and higher sputum eosinophilia; obese asthmatics had worse asthma control and quality of life, and tended to have lower sputum eosinophils; asthmatics with GERD showed worse quality of life. In multivariate analysis, obesity was the only independent factor associated with poor asthma control (OR 4.9), while CRSwNP was the only independent factor associated with airway eosinophilia (OR 16.2). Lower lung function was associated with the male gender and longer duration of asthma (OR 3.9 and 5.1, respectively) and showed a trend for the association with nasal polyps (OR 2.9, p = 0.06). CONCLUSION: Our study suggests that coexisting comorbidities are associated with different features of SA.

The beclomethasone anti-inflammatory effect occurs in cell/mediator-dependent manner and is additively enhanced by formoterol: NFkB, p38, PKA analysis.

AIMS: Beclomethasone/formoterol (BDP/FOR) has been reported to be more effective than its separate components in airway disease control and in airway inflammation improvement. However, BDP/FOR effects on cytokine-induced inflammation in structural cells have not been described and whether these effects occur in a cell- and mediator-dependent manner has not been fully elucidated. We sought to evaluate BDP and/or FOR effects on endothelial ICAM-1, E-selectin, IL-8 and on bronchial epithelial ICAM-1 and IL-8. Specific intracellular signaling pathways were also investigated. MATERIALS AND METHODS: Surface adhesion molecule expression and IL-8 release induced by TNF-alpha were measured by ELISA. Intracellular signaling pathways were investigated by a) EMSA and Western blot analysis to evaluate NF-κB DNA-binding and MAPK-p38 phosphorylation; b) PDTC/SB203580 as NF-κB/p38 inhibitors; c) forskolin/H-89 as PKA activator/inhibitor. KEY FINDINGS: BDP/FOR additively reduced endothelial E-selectin and IL-8 as well as bronchial epithelial ICAM-1 and IL-8. BDP/FOR and SB203580 showed the highest inhibitory effect on epithelial IL-8, whereas endothelial ICAM-1 was never affected by BDP/FOR and PDTC. TNF-alpha-induced NF-κB DNA-binding and MAPK-p38 phosphorylation were not influenced by BDP/FOR. Forskolin mimicked FOR effects; H-89 partially reversed the BDP/FOR inhibition in a mediator-dependent manner. SIGNIFICANCE: The BDP/FOR inhibition degree was related to the inflammatory mediator- and cell-type considered. FOR additively enhanced BDP effects by partially involving both dependent- and independent-PKA mechanisms. Our results might contribute to highlight the strong relationship between specific molecular pathways and different sensitivity to the corticosteroid/ß2-agonist effects and to clarify the molecular mechanisms underlying the BDP/FOR anti-inflammatory activity in vivo.

Inhalation therapy in the next decade: Determinants of adherence to treatment in asthma and COPD.

Proceedings of the European Seminars in Respiratory Medicine course, Inhalation therapy in the next decade: Determinants of adherence to treatment in asthma and COPD, held in Taormina, Italy, on 3-4 March, 2017.

Mediators of angiogenesis and fibrosis in IgG4-related disease.

IgG4-related disease (IgG4-RD) is a rare fibro-inflammatory condition that can affect almost any organ, characterized by tumefactive lesions and often by eosinophilia and elevated serum IgG4 concentrations. The aim of the study is to analyze in IgG4-RD patients the serum levels of a group of cytokines and growth factors potentially involved in the regulation of fibrotic processes. In the sera of 12 patients affected by IgG4-RD and of 15 normal healthy subjects (NHS), pro-fibrogenic mediators (TGF-ß1 and periostin) and pro- (VEGF and angiogenin-1) and anti- (endostatin and thrombospondin-1) angiogenic mediators were measured by sandwich enzyme immunoassay. Among mediators regulating fibrosis and angiogenesis, endostatin levels were significantly higher in IgG4-RD patients compared to NHS (p < 0.0001). No differences in the levels of TGF-ß1, periostin, VEGF, angiogenin-1 and thrombospondin-1 were observed between groups. Our study suggests that among the mediators mainly involved in fibrosis and angiogenesis endostatin might play a role in the pathogenetic processes of IgG4-RD.

Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells.

BACKGROUND: Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar. METHODS: Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient. RESULTS: Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action. CONCLUSIONS: GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.

The effect of high glucose on the inhibitory action of C21, a selective AT2R agonist, of LPS-stimulated tissue factor expression in human mononuclear cells.

BACKGROUND: Intimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively). RESULTS: HG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG. CONCLUSIONS: HG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathways.

Asthma Control and Airway Inflammation in Patients with Eosinophilic Granulomatosis with Polyangiitis.

BACKGROUND: Eosinophilic granulomatosis with polyangiitis (EGPA) is a systemic necrotizing vasculitis that occurs in patients with asthma, nasal disease, blood and tissue eosinophilia, and extrapulmonary manifestations. OBJECTIVE: The aim of our study was to assess the clinical, functional, and inflammatory status of upper and lower airways in 37 patients with EGPA, examined 6.4 ± 4.7 years after diagnosis, when they were in partial or complete remission from systemic involvement while on treatment with low-dose oral corticosteroids as maintenance therapy. METHODS: All patients performed spirometry and were assessed for bronchial hyperreactivity, sputum eosinophilia, and fractional exhaled nitric oxide; asthma control was evaluated according to the Global Initiative for Asthma (GINA) guidelines and the Asthma Control Test. Markers of systemic disease were compared with the data available at diagnosis. Nasal involvement was evaluated by using the Sino-Nasal Outcome Test, nasal endoscopy, and nasal cytology. The impact on the quality of life was evaluated by using generic (36-item short form health survey) and organ-specific questionnaires. RESULTS: At the time of the study visit, almost all patients were receiving low-dose oral corticosteroids and immunomodulating drugs, but only 50% were being treated with inhaled corticosteroids. Although low systemic disease activity was documented in the large majority of patients, poorly controlled asthma and rhinosinusitis with eosinophilic airway inflammation were demonstrated in almost all patients. A significant correlation was found between sputum and blood eosinophilia and between fractional exhaled nitric oxide and asthma control. The 36-item short form health survey questionnaire results significantly correlated with the Sino-Nasal Outcome Test but not with the Asthma Control Test. CONCLUSIONS: Systemic treatment controls systemic involvement in EGPA, but not asthma and nasal diseases, which negatively affects patients' quality of life.

Monocytes/macrophages activation contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques.

BACKGROUND: Gamma-glutamyltransferase (GGT) is a well-established independent risk factor for cardiovascular mortality related to atherosclerotic disease. Four GGT fractions have been identified in plasma, but only b-GGT fraction accumulates in atherosclerotic plaques, and correlates with other histological markers of vulnerability. The present study was aimed to evaluate whether macrophagic lineage cells may provide a source of b-GGT within the atherosclerotic plaque. METHODS: GGT expression and release were studied in human monocytes isolated from peripheral blood of healthy donors. The growth factors GM-CSF and M-CSF were used to induce differentiation into M1-like and M2-like macrophages, respectively. Plaque GGT was investigated in tissue samples obtained from patients undergoing carotid endoarterectomy. RESULTS: We found that M1-like macrophages express higher levels of GGT as compared to M2-like, and that both monocytes and M1-like macrophages-but not M2-like-are able to release the b-GGT fraction upon activation with pro-inflammatory stimuli. Western blot analysis of b-GGT extracted from plaques confirmed the presence of a GGT immunoreactive peptide coincident with that of macrophages. CONCLUSIONS: Our data indicate that macrophages characterized by a pro-inflammatory phenotype may contribute to intra-plaque accumulation of b-GGT, which in turn may play a role in the progression of atherosclerosis by modulating inflammatory processes and favouring plaque instability.

Can Sputum Eosinophilia Be a Constant Feature in Severe Refractory Asthmatics? A 3-Year Longitudinal Study.

In difficult-to-treat asthmatics, uncontrolled despite a high level of therapy and followed for 3 years with a mean number of sputum samples/patient = 10, sputum eosinophilia (≥3%) was observed in 87% of all sputum samples. Persistent sputum eosinophilia is a characteristic of severe uncontrolled asthma.

Sputum eosinophilia is a determinant of FEV1 decline in occupational asthma: results of an observational study.

OBJECTIVE: To evaluate the potential determinants of forced expiratory volume in 1 s (FEV1) decline in workers with occupational asthma (OA) still exposed to the causative agent. We hypothesised that sputum eosinophilia might be a predictor of poor asthma outcome after diagnosis. SETTING, DESIGN AND PARTICIPANTS: In a specialistic clinical centre of the University Hospital of Pisa, we studied 39 participants (28 M, 11 F) diagnosed as having OA, routinely followed up between 1990 and 2009. They were a subgroup of 94 participants diagnosed as affected by OA in that period: 9 had been removed from work at the diagnosis, 21 were excluded for having ceased occupational exposure after few months from diagnosis, and 25 were lost at the follow-up or had no acceptable sputum measurements at the diagnosis. Estimates of the decline in FEV1 were obtained by means of simple regression analysis during the period of occupational exposure after diagnosis. Logistic regression was used to analyse the effects of factors (baseline FEV1 and sputum inflammatory cells, duration and type of exposure) that may potentially influence FEV1 decline. RESULTS: At follow-up (5.7+3.7 years), most participants were still symptomatic despite inhaled corticosteroids (ICS) treatment and had their occupational exposure reduced. Participants with higher sputum eosinophils (>3%) at baseline had a significantly greater decline of FEV1 (-52.5 vs -18.6 mL/year, p=0.012). Logistic regression showed that persistent exposure and sputum eosinophilia were significantly associated with a greater decline in FEV1 (OR 11.5, 95% CI 1.8 to 71.4, p=0.009 and OR 6.7, 95% CI 1.1 to 41.7, p= 0.042, respectively). CONCLUSIONS: Sputum eosinophilia at diagnosis, together with the persistence of occupational exposure during follow-up, may contribute to a greater decline in FEV1 in patients with OA still at work. Further long-term studies are required as to whether intensive ICS treatment may be beneficial for patients with OA and increase ad eosinophilic inflammation.

Neutrophilic Bronchial Inflammation Correlates with Clinical and Functional Findings in Patients with Noncystic Fibrosis Bronchiectasis.

Background. Neutrophilic bronchial inflammation is a main feature of bronchiectasis, but not much is known about its relationship with other disease features. Aim. To compare airway inflammatory markers with clinical and functional findings in subjects with stable noncystic fibrosis bronchiectasis (NCFB). Methods. 152 NFCB patients (62.6 years; females: 57.2%) underwent clinical and functional cross-sectional evaluation, including microbiologic and inflammatory cell profile in sputum, and exhaled breath condensate malondialdehyde (EBC-MDA). NFCB severity was assessed using BSI and FACED criteria. Results. Sputum neutrophil percentages inversely correlated with FEV1 (P < 0.0001; rho = -0.428), weakly with Leicester Cough Questionnaire score (P = 0.068; rho = -0.58), and directly with duration of the disease (P = 0.004; rho = 0.3) and BSI severity score (P = 0.005; rho = 0.37), but not with FACED. Sputum neutrophilia was higher in colonized subjects, P. aeruginosa colonized subjects showing greater sputum neutrophilia and lower FEV1. Patients with ≥3 exacerbations in the last year showed a significantly greater EBC-MDA than the remaining patients. Conclusions. Sputum neutrophilic inflammation and biomarkers of oxidative stress in EBC can be considered good biomarkers of disease severity in NCFB patients, as confirmed by pulmonary function, disease duration, bacterial colonization, BSI score, and exacerbation rate.

Compound 21, a selective angiotensin II type 2 receptor agonist, downregulates lipopolysaccharide-stimulated tissue factor expression in human peripheral blood mononuclear cells.

Intricate interrelationships connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally active angiotensin II, a proinflammatory agent with direct TF-stimulating properties mediated by the angiotensin II type 1 receptor (AT1R)s. However, angiotensin II also stimulates angiotensin II type 2 receptor (AT2R)s and they may as well contribute to TF expression, a possibility in need of further evaluation. We investigated the effect of C21, a highly specific AT2R agonist, on TF antigen (ELISA), procoagulant activity (PCA, one-stage clotting assay) and TF-mRNA (real-time PCR) in peripheral blood mononuclear cell (PBMC)s activated by lipopolysaccharide (LPS), a pro-inflammatory and procoagulant stimulus. C21 downregulated LPS-stimulated TF antigen, PCA and TF mRNA, an effect abolished by PD123 319, a selective AT2R antagonist, and left unchanged by omesartan, a selective AT1R antagonist. PD123 319 per se did not affect LPS-induced TF expression while omesartan inhibited and BAY 11-7082, a specific NFκB inhibitor, abolished endotoxin-activated procoagulant activity (PCA). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated PBMCs, a finding consistent with the existence in PBMCs of AT2Rs whose stimulation attenuates inflammation-mediated procoagulant responses. The data open insofar unexplored and potentially relevant facets to our understanding of the complex links connecting angiotensin II to inflammation and coagulation.

γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells.

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.

Angiotensin II induces the generation of procoagulant microparticles by human mononuclear cells via an angiotensin type 2 receptor-mediated pathway.

INTRODUCTION: Microparticles are small vesicles shed by cells upon activation and during apoptosis which participate in physiologically relevant phenomena, including blood coagulation. Intracellular calcium mobilization is one of the mechanisms of microparticle generation during cell activation. Because the renin-angiotensin system has been proposed as a link between hypertension and increased thrombotic risk, we investigated whether angiotensin II upregulates the generation of procoagulant microparticles by human mononuclear cells. MATERIALS AND METHODS: Human mononuclear cells were exposed to angiotensin II for 15min. Intracellular calcium concentration was assessed by a Fluo 4 based kit. The supernatants were analyzed for both microparticle content, with a commercially available kit based on phosphatidylserine analysis, and microparticle-associated tissue factor, with a one-stage clotting assay. RESULTS: Intracellular calcium concentration is increased upon exposure of mononuclear cells to angiotensin II. Incubation with angiotensin II stimulates microparticles release; microparticle-associated tissue factor is also upregulated. The effect is inhibited by an angiotensin receptor type 2 antagonist (PD123319) and not by two angiotensin type 1 antagonists (Losartan and Olmesartan). CONCLUSIONS: Angiotensin receptor 2-mediated upregulation of tissue factor-bearing, procoagulant microparticle generation represents a novel mechanism linking the renin-angiotensin system to thrombosis.

Baseline airway inflammation may be a determinant of the response to ozone exposure in asthmatic patients.

CONTEXT: It is well known that ozone exposure decreases lung function and increases airway neutrophilia, but large variability has been observed among asthmatic patients. OBJECTIVE: To find possible predictors of functional and inflammatory airway response to ozone in asthmatic patients. MATERIALS AND METHODS: We studied 120 patients with mild-to-moderate asthma, randomly exposed to either air or ozone (0.3 ppm for 2 h) in a challenge chamber. Symptoms and pulmonary function test (PFT) were measured before and immediately after exposure. Six hours after exposure, induced sputum was collected. Patients were evaluated according to their functional (FEV1 responders) or neutrophilic (neutrophil responders) response to ozone. We considered, as possible predictors of response: age, baseline FEV1, previous treatment with inhaled corticosteroids (ICS), baseline sputum neutrophils, baseline sputum eosinophils, methacholine responsiveness, atopy and smoking habit. RESULTS: FEV1 responders had lower baseline FEV1, and a lower percentage of these had received ICS treatment. Neutrophil responders were younger, with lower baseline sputum inflammation and greater methacholine responsiveness. These results were confirmed by multivariate logistic analysis. DISCUSSION AND CONCLUSION: Patients not previously treated with ICS and patients with lower FEV1 are more prone to functional response to ozone. Lower baseline airway inflammation and greater bronchial hyperresponsiveness may predict neutrophilic airway response to ozone in asthmatic patients. Thus, determinants of functional and inflammatory responses to ozone are different.