Cell-derived extracellular vesicles as potential effectors of thromboinflammation in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is associated with both local and systemic inflammation. Severe acute exacerbations of COPD (AE-COPD, defined as acute worsening in respiratory symptoms, beyond the usual day-by-day variability and requiring hospital admission) are associated with an excess of cardiovascular morbidity and mortality. It is conceivable that systemic inflammation is involved in this increased risk. Moreover, it is well known that inflammation and coagulation are closely related. The detrimental interplay between coagulation and inflammation is now referred to as thromboinflammation. Interestingly, many of the major cardiovascular events (myocardial infarction, stroke, pulmonary embolism) associated with severe AE-COPD recognize underlying thromboinflammatory mechanisms. Extracellular vesicles (EV) are small membranous vesicles, released by all human cells, both constitutively and upon specific stimuli, including inflammation, and are involved in intercellular communication. Moreover, EV seem to represent ideal players in promoting blood coagulation and thrombosis, since they can expose on their outer surface tissue factor (TF), the physiological initiator of coagulation in vivo. Even though there are some data about thromboinflammatory mechanisms in COPD, no studies have so far focused on EV. The aim of this PhD project is therefore to investigate the potential thromboinflammatory effects of circulating EV from COPD patients. The research questions are the following: 1) can circulating EV exert a significant pro-thrombotic activity in COPD patients? 2) May EV represent pro-inflammatory effectors of the disease, through the stimulation of specific cell targets? EV-associated TF concentration (EV-TF), measured through a one-stage clotting assay in which TF availability represents the limiting factor for the reaction, was used to evaluate plama-derived EV pro-thrombotic activity. To investigate EV-associated pro-inflammatory activity, EV were incubated with the bronchial cell line 16HBE and the EV-induced production of interleukin-8 (IL-8) and C-C motive chemokine ligand-2 (CCL-2). We enrolled 3 groups of subjects: 1) patients with severe AE-COPD, evaluated twice: at hospital admission (V0) and 8 weeks later (V1); 2) patients with stable COPD (sCOPD, no exacerbations in the last 12 months); 3) control subjects without COPD. The final population included 93 subjects (30 AE-COPD, 50 sCOPD and 13 controls). In AE-COPD we found significantly higher EV-TF concentration at V0 than at V1. Moreover, EV-TF still remains higher at both V0 and V1 in AE-COPD than in sCOPD and in controls. We found the same statistically significant results for IL-8 and CCL-2, whose levels were higher at V0 than at V1, and higher in AE-COPD than in sCOPD and in controls. Last, we conducted a follow-up study in a subgroup of COPD patients (14 AE-COPD and 21 sCOPD), who were re-evaluated at 6 months. We found that EV-TF, IL-8 and CCL-2 all remained significantly higher in AE-COPD than in sCOPD at 6 months. Taken together, these results confirm that EV can be involved in the thromboinflammatory events that are associated with AE-COPD and that could be responsible for the augmented, long lasting cardiovascular risk which characterizes severe AE-COPD.