Enhanced prothrombotic and proinflammatory activity of circulating extracellular vesicles in acute exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are associated with a high rate of cardiovascular events. Thromboinflammation (the interplay between coagulation and inflammation) is probably involved in these events. Extracellular vesicles (EV) increase during AE-COPD, but their role in thromboinflammation in COPD is still unknown. We investigated EV-associated prothrombotic and proinflammatory activity in COPD. METHODS: Patients with AE-COPD, stable COPD (sCOPD) and age- and sex-matched subjects (controls) were enrolled. AE-COPD patients were evaluated at hospital admission and 8 weeks after discharge (recovery; longitudinal arm). In a cross-sectional arm, AE-COPD were compared with sCOPD and controls. EV-mediated prothrombotic activity was tested by measuring the concentration of EV-associated phosphatidylserine, as assessed by a prothrombinase assay, and tissue factor, as assessed by a modified one-stage clotting assay (EV-PS and EV-TF, respectively). Synthesis of interleukin-8 (IL-8) and C-C motif chemokine ligand-2 (CCL-2) by cells of the human bronchial epithelial cell line 16HBE incubated with patients' EV was used to measure EV-mediated proinflammatory activity. RESULTS: Twenty-five AE-COPD (median age [interquartile range] 74.0 [14.0] years), 31 sCOPD (75.0 [9.5] years) and 12 control (67.0 [3.5] years) subjects were enrolled. In the longitudinal arm, EV-PS, EV-TF, IL-8 and CCL-2 levels were all significantly higher at hospital admission than at recovery. Similarly, in the cross-sectional arm, EV-PS, EV-TF and cytokines synthesis were significantly higher in AE-COPD than in sCOPD and controls. CONCLUSIONS: EV exert prothrombotic and proinflammatory activities during AE-COPD and may therefore be effectors of thromboinflammation, thus contributing to the higher cardiovascular risk in AE-COPD.

Extracellular Vesicles in Pulmonary Hypertension: A Dangerous Liaison?

The term pulmonary hypertension (PH) refers to different conditions, all characterized by increased pressure and resistance in the pulmonary arterial bed. PH has a wide range of causes (essentially, cardiovascular, pulmonary, or connective tissue disorders); however, idiopathic (i.e., without a clear cause) PH exists. This chronic, progressive, and sometimes devastating disease can finally lead to right heart failure and eventually death, through pulmonary vascular remodeling and dysfunction. The exact nature of PH pathophysiology is sometimes still unclear. Extracellular vesicles (EVs), previously known as apoptotic bodies, microvesicles, and exosomes, are small membrane-bound vesicles that are generated by almost all cell types and can be detected in a variety of physiological fluids. EVs are involved in intercellular communication, thus influencing immunological response, inflammation, embryogenesis, aging, and regenerative processes. Indeed, they transport chemokines, cytokines, lipids, RNA and miRNA, and other biologically active molecules. Although the precise functions of EVs are still not fully known, there is mounting evidence that they can play a significant role in the pathophysiology of PH. In this review, after briefly recapping the key stages of PH pathogenesis, we discuss the current evidence on the functions of EVs both as PH biomarkers and potential participants in the distinct pathways of disease progression.

Do Circulating Extracellular Vesicles Strictly Reflect Bronchoalveolar Lavage Extracellular Vesicles in COPD?

Cell-derived extracellular vesicles (EVs) found in the circulation and body fluids contain biomolecules that could be used as biomarkers for lung and other diseases. EVs from bronchoalveolar lavage (BAL) might be more informative of lung abnormalities than EVs from blood, where information might be diluted. To compare EVs' characteristics in BAL and blood in smokers with and without COPD. Same-day BAL and blood samples were obtained in 9 nonsmokers (NS), 11 smokers w/o COPD (S), and 9 with COPD (SCOPD) (FEV1: 59 ± 3% pred). After differential centrifugation, EVs (200-500 nm diameter) were identified by flow cytometry and labeled with cell-type specific antigens: CD14 for macrophage-derived EVs, CD326 for epithelial-derived EVs, CD146 for endothelial-derived EVs, and CD62E for activated-endothelial-derived EVs. In BAL, CD14-EVs were increased in S compared to NS [384 (56-567) vs. 172 (115-282) events/µL; p = 0.007] and further increased in SCOPD [619 (224-888)] compared to both S (p = 0.04) and NS (p < 0.001). CD326-EVs were increased in S [760 (48-2856) events/µL, p < 0.001] and in SCOPD [1055 (194-11,491), p < 0.001] when compared to NS [15 (0-68)]. CD146-EVs and CD62E-EVs were similar in the three groups. In BAL, significant differences in macrophage and epithelial-derived EVs can be clearly detected between NS, S and SCOPD, while these differences were not found in plasma. This suggests that BAL is a better medium than blood to study EVs in lung diseases.

The Emerging Role of Extracellular Vesicles Detected in Different Biological Fluids in COPD.

The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by complex cellular and molecular mechanisms, not fully elucidated so far. It involves inflammatory cells (monocytes/macrophages, neutrophils, lymphocytes), cytokines, chemokines and, probably, new players yet to be clearly identified and described. Chronic local and systemic inflammation, lung aging and cellular senescence are key pathological events in COPD development and progression over time. Extracellular vesicles (EVs), released by virtually all cells both as microvesicles and exosomes into different biological fluids, are involved in intercellular communication and, therefore, represent intriguing players in pathobiological mechanisms (including those characterizing aging and chronic diseases); moreover, the role of EVs as biomarkers in different diseases, including COPD, is rapidly gaining recognition. In this review, after recalling the essential steps of COPD pathogenesis, we summarize the current evidence on the roles of EVs collected in different biological mediums as biomarkers in COPD and as potential players in the specific mechanisms leading to disease development. We will also briefly review the data on EV as potential therapeutic targets and potential therapeutic agents.

Pirfenidone for Idiopathic Pulmonary Fibrosis and Beyond.

Pirfenidone (PFD) slows the progression of idiopathic pulmonary fibrosis (IPF) by inhibiting the exaggerated fibrotic response and possibly through additional mechanisms, such as anti-inflammatory effects. PFD has also been evaluated in other fibrosing lung diseases. Myocardial fibrosis is a common feature of several heart diseases and the progressive deposition of extracellular matrix due to a persistent injury to cardiomyocytes may trigger a vicious cycle that leads to persistent structural and functional alterations of the myocardium. No primarily antifibrotic medications are used to treat patients with heart failure. There is some evidence that PFD has antifibrotic actions in various animal models of cardiac disease and a phase II trial on patients with heart failure and preserved ejection fraction has yielded positive results. This review summarises the evidence about the possible mechanisms of IPF and modulation by PFD, the main results about IPF or non-IPF interstitial pneumonias and also data about PFD as a potential protective cardiac drug.

A novel prothrombotic role of proprotein convertase subtilisin kexin 9: the generation of procoagulant extracellular vesicles by human mononuclear cells.

BACKGROUND: Proprotein convertase subtilisin kexin 9 (PCSK9) is a serin protease synthesized mainly in the liver that binds the receptor of low-density lipoprotein and promotes its degradation in lysosomes. PCSK9 is considered a promising target for the development of new therapies for the treatment of hypercholesterolemia and related cardiovascular diseases. Extracellular vesicles represent a heterogeneous population of vesicles, ranging in size between 0.05 and 1 µm involved in numerous pathophysiological processes, including blood coagulation. We investigated whether PCSK9 stimulation induces the release of procoagulant extracellular vesicles from human mononuclear cells (PBMCs) and THP-1 cells. METHODS AND RESULTS: PBMCs and THP-1 cells were stimulated whit PCSK9, the generation of EV was assessed by the prothrombinase assay and by cytofluorimetric analysis. EV-associated tissue factor activity was assessed by a one-stage clotting assay. PCSK9 induced an increase in extracellular generation by PBMCs and THP-1 cells as well as an increase in extracellular vesicle-associated tissue factor. Pre-treatment with inhibitors of the toll like receptor, TLR4 (C34), and of NF-κB signaling (BAY 11-7082), downregulated PCSK9-induced extracellular vesicle generation and of extracellular- bound tissue factor. Similar effect was obtained by an anti-PCSK9 human-monoclonal antibody. CONCLUSIONS: PCSK9-mediated generation of procoagulant EV could contribute to increase the prothrombotic status in patients with cardiovascular diseases.

Circulating Extracellular Vesicles Are Associated with Disease Severity and Interleukin-6 Levels in COPD: A Pilot Study.

Chronic obstructive pulmonary disease (COPD) is a complex condition in which systemic inflammation plays a role in extrapulmonary manifestations, including cardiovascular diseases: interleukin (IL)-6 has a role in both COPD and atherogenesis. The 2011 GOLD document classified patients according to FEV1, symptoms, and exacerbations history, creating four groups, from A (less symptoms/low risk) to D (more symptoms/high risk). Extracellular vesicles (EV) represent potential markers in COPD: nevertheless, no studies have explored their value in association to both disease severity and inflammation. We conducted a pilot study to analyze circulating endothelial-(E) and monocyte-derived (M) EV levels in 35 COPD patients, who were grouped according to the 2011 GOLD document; the relationship between EV and plasmatic markers of inflammation was analyzed. We found a statistically significant trend for increasing EEV, MEV, IL-6, from group A to D, and a significant correlation between EEV and IL-6. The associations between both EEV and MEV and disease severity, and between EEV and IL-6, suggest a significant interplay between pulmonary disease and inflammation, with non-respiratory cells (endothelial cells and monocytes) involvement, along with the progression of the disease. Thus, EV might help identify a high-risk population for extrapulmonary events, especially in the most severe patients.

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.

C-C motive chemokine ligand 2 and thromboinflammation in COVID-19-associated pneumonia: A retrospective study.

PURPOSE: A derangement of the coagulation process and thromboinflammatory events has emerged as pathologic characteristics of severe COVID-19, characterized by severe respiratory failure. CC motive chemokine ligand 2 (CCL2), a chemokine originally described as a chemotactic agent for monocytes, is involved in inflammation, coagulation activation and neoangiogenesis. We investigated the association of CCL2 levels with coagulation derangement and respiratory impairment in patients with COVID-19. METHODS: We retrospectively evaluated 281 patients admitted to two hospitals in Italy with COVID-19. Among them, CCL2 values were compared in different groups (identified according to D-dimer levels and the lowest PaO2/FiO2 recorded during hospital stay, P/Fnadir) by Jonckheere-Terpstra tests; linear regression analysis was used to analyse the relationship between CCL2 and P/Fnadir. We performed Mann-Whitney test and Kaplan-Meier curves to investigate the role of CCL2 according to different clinical outcomes (survival and endotracheal intubation [ETI]). RESULTS: CCL2 levels were progressively higher in patients with increasing D-dimer levels and with worse gas exchange impairment; there was a statistically significant linear correlation between log CCL2 and log P/Fnadir. CCL2 levels were significantly higher in patients with unfavourable clinical outcomes; Kaplan-Meier curves for the composite outcome death and/or need for ETI showed a significantly worse prognosis for patients with higher (> median) CCL2 levels. CONCLUSIONS: CCL2 correlates with both indices of activation of the coagulation cascade and respiratory impairment severity, which are likely closely related in COVID-19 pathology, thus suggesting that CCL2 could be involved in the thromboinflammatory events characterizing this disease.

Expression Analysis of Muscle-Specific miRNAs in Plasma-Derived Extracellular Vesicles from Patients with Chronic Obstructive Pulmonary Disease.

MicroRNAs (miRNAs) are a class of short non-coding RNAs involved in the regulation of gene expression and the control of several cellular processes at physiological and pathological levels. Furthermore, extracellular vesicles (EV), which are small membrane-bound vesicles secreted by cells in the extracellular environment, contain functional miRNAs. The remarkable deregulation of many miRNAs has been demonstrated in respiratory diseases. Among them, miR-206, miR-133a-5p, and miR-133a-3p are striated muscle-specific miRNAs (myo-miRNA), related to skeletal muscle dysfunction, one of the commonest systemic manifestations in patients with chronic obstructive pulmonary disease (COPD). Nevertheless, their circulating expression in COPD patients is not demonstrated. For these reasons, we performed a pilot study to analyze the expression profiles of myo-miRNAs in plasma-derived EV from patients with COPD. We analyzed the expression profiles of selected myo-miRNAs in plasma-derived EV from COPD. Receiver operating characteristic analyses were carried out to evaluate whether selected plasma miRNAs were able to discriminate between different groups of COPD patients. We found EV-embedded myo-miRNAs in the bloodstream of COPD patients. Specifically, miR-206, miR-133a-5p and miR-133a-3p were significantly upregulated in group B patients. Receiver operating characteristic analyses of the combination of these selected miRNAs showed their high capacity to discriminate group B from other COPD patients. Our data provide evidence that myo-miRNA are present in EV in the plasma of COPD patients and their expression (miR-206, miR-133a-5p, and miR-133a-3p) can discriminate group B from group C patients. The future analysis of a larger number of patients should allow us to obtain more refined correlations.

Pulmonary embolism: yet another cause of hypoxaemic respiratory failure in COVID-19.

Pulmonary embolism represents an overlooked cause of worsening respiratory failure in COVID-19. A regular bedside evaluation for atypical features like pleuritic chest pain or pleural effusion could help identify suspected cases for appropriate management. https://bit.ly/3bbBPqZ.

Fluticasone furoate, umeclidinium bromide, and vilanterol as a combination therapy for chronic obstructive pulmonary disease.

Introduction: Triple therapy with two bronchodilators (LABA plus LAMA) and an inhaled corticosteroid (ICS) is recommended for patients suffering from severe chronic obstructive pulmonary disease (COPD). Areas covered: All 12-52 week-long studies comparing triple therapy with umeclidinium (UM) added to either fluticasone furoate/vilanterol (FF/VI) or fluticasone propionate/salmeterol (FP/SAL) vs. other comparators in COPD patients of group B or D (2011 GOLD classification) were considered. When UM was added to ICS/LABA with separate devices or within a single device, triple combination was more effective than comparators (usually, ICS/LABA combinations) regarding improvements to pulmonary function, symptoms, quality of life and, in the longer studies, rate of moderate-severe exacerbations. The IMPACT study (a large trial comparing UM/FF/VI with both FF/VI and UM/VI combinations) showed that triple therapy had a greater effect compared to dual therapies in reducing the rate of moderate-severe exacerbations, improving trough FEV1 and improving quality of life. The safety profile was good, without excess cardiovascular effects or pneumonia, however, the presence of comorbidities was frequent. Expert commentary: UM/FF/VI combination represents a good option for severe COPD patients who remain symptomatic and/or with frequent exacerbations despite dual therapies. Once daily administration with a simple and effective device may increase adherence and efficacy of the treatment.

Cell-derived microparticles and the lung.

Cell-derived microparticles are small (0.1-1 µm) vesicles shed by most eukaryotic cells upon activation or during apoptosis. Microparticles carry on their surface, and enclose within their cytoplasm, molecules derived from the parental cell, including proteins, DNA, RNA, microRNA and phospholipids. Microparticles are now considered functional units that represent a disseminated storage pool of bioactive effectors and participate both in the maintenance of homeostasis and in the pathogenesis of diseases. The mechanisms involved in microparticle generation include intracellular calcium mobilisation, cytoskeleton rearrangement, kinase phosphorylation and activation of the nuclear factor-κB. The role of microparticles in blood coagulation and inflammation, including airway inflammation, is well established in in vitro and animal models. The role of microparticles in human pulmonary diseases, both as pathogenic determinants and biomarkers, is being actively investigated. Microparticles of endothelial origin, suggestive of apoptosis, have been demonstrated in the peripheral blood of patients with emphysema, lending support to the hypothesis that endothelial dysfunction and apoptosis are involved in the pathogenesis of the disease and represent a link with cardiovascular comorbidities. Microparticles also have potential roles in patients with asthma, diffuse parenchymal lung disease, thromboembolism, lung cancer and pulmonary arterial hypertension.

An observation of prescription behaviors and adherence to guidelines in patients with COPD: real world data from October 2012 to September 2014.

INTRODUCTION: GOLD guideline recommendations are currently the "gold standard" for the treatment of COPD patients. OBJECTIVES: The objective of this analysis was to evaluate compliance with GOLD guidelines in managing COPD patients' treatment by general practitioners (GPs) and pulmonologists. Since inhaled corticosteroid (ICS) use is defined as inappropriate in mild and moderate COPD patients, special attention was paid to ICS therapy use in these classes. METHODS: The study was based on the Italian GP database IMS Health Longitudinal Patient Database (IMS Health LPD) and on the Patient Analyzer specialist IMS Health database. The observed cohort included all patients with a diagnosis of COPD, aged 40 years or more, with at least one ATC R03 class prescription, visited by GPs and pulmonologists during four timeframes: October 2012 - March 2013 (cohort 1), April 2013 - September 2013 (cohort 2), October 2013 - March 2014 (cohort 3); April 2014 - September 2014 (cohort 4). Patients were classified into disease severity groups following 2008 GOLD guidelines, based on FEV1 value. RESULTS: Cohorts were quite similar in size (about two thousand patients per cohort). Pulmonologists visited more severe patients than GPs. About 50% of GPs' mild and moderate patients received treatments containing inhaled corticosteroids. Pulmonologists were more adherent to guidelines, with smaller percentages of mild patients treated with therapies containing ICS (ranging from 19.0% to 30.1%). An improvement in adherence was observed during the four time periods, with a decrease in the use of therapies containing ICS in mild and moderate patients. In absolute terms, it emerged that GPs more often prescribe ICS improperly to patients in the mild and moderate severity classes than pulmonologists. CONCLUSION: Real world data indicate that adherence to GOLD guidelines is only partially met by GPs in their general practice and shows higher prescription appropriateness by pulmonologists.

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.

Acute administration of bronchodilators on exercise tolerance in treated COPD patients.

Exercise intolerance is a major feature in patients with Chronic Obstructive Pulmonary Disease (COPD). Bronchodilators increase endurance time (ET) and reduce dynamic hyperinflation (DH). We evaluated whether a single-dose of salbutamol/ipratropium + flunisolide (BD+ICS), added on top of the regular treatment, may improve ET in COPD patients. In a single-blind randomized crossover pilot trial, nebulised BD+ICS or placebo (PL) was administered 30 min before a constant load cardiopulmonary test, in 22 moderate-to-severe COPD patients (FEV1: 53.9% pred). ET was the primary outcome measured. BD+ICS did not improve ET or VO2 peak with respect to PL. BD+ICS increased pre-test FEV1 and pre-test Inspiratory Capacity but did not modify DH. In a retrospective analysis, patients were divided in Improvers (N=11) and Non-Improvers (N=11) according to the difference in ET between BD+ICS and PL (> 25 s). Improvers had a worst BODE index, a higher static hyperinflation and poorer Vd/Vt ratio at peak of exercise with respect to Non-Improvers. Improvers only had a significant increase from BD+ICS on pre-test FEV1 and IC. In conclusion, although a single-dose BD+ICS did not improve ET in COPD patients under regular treatment, a subgroup of more severe patients may have some benefit from that.