Proteomic analysis reveals procoagulant properties of cigarette smoke‐induced extracellular vesicles

Abstract

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. ARTICLE HISTORY

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells

“…However, very few studies have analyzed the RNA cargo of EVs generated after ETS. Recently, Stassen and his group showed that tissue factor procoagulants are released from bronchial epithelial cells in response to contact with cigarette smoke extract through an EVs mechanism, suggesting a possible function of EVs after smoke exposure [26,27].…”

Cell Type- and Exposure-Specific Modulation of CD63/CD81-Positive and Tissue Factor-Positive Extracellular Vesicle Release in response to Respiratory Toxicants

“…We have previously shown that cigarette smoke extract (CSE) increases the amount of small (80-250 nm) CD63 + CD81 + EVs released by bronchial epithelial cells [18]. These CSE-induced EVs were enriched in tissue factor (TF) compared to EVs secreted by unexposed cells [23]. Thus, they likely reflect epithelial activation and damage.…”

Extracellular Vesicles in Cardiovascular Diseases: Alternative Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers

“…EV proteins have also been associated with CVD risk factors, such as smoking and obesity. In an in vitro study with bronchial epithelium cells, EVs derived from epithelial cells that were exposed to cigarette smoke extract were shown to contain higher levels of pro-coagulant proteins, including tissue factor [83]. In fact, EVs present in human blood contain several coagulation-related proteins and therefore, may contribute to the increased risk of a thrombotic event resulting in MI or stroke [24,43,84].…”