Appendix1. Summary of the reviewed articles describing associations between the internal/external environment (ie, air pollution, smoking, alcohol consumption, obesity, oxidative stress, and aging) and extracellular vesicle targets (origin, content, and count)

First Author, year / Internal/external environment / Design / N / Population / Investigation method / Target / Results
Emmerechts, 2012 / Air Pollution / Cohort Study / 233 / Type 1 and 2 diabetic patients / Flowcytometry (FACSCantoll) / Origin / Day of blood sampling levels of PM2.5were associated with the amount of circulating MV: for a 10µg/m3 increase in PM2.5, blood platelet-derived MVs were decreased with 57% (95% CI: -78% to -16%) and annexin V-binding derived MVs decreased 74%; (95% CI: -85% to -56%).
Positive correlations between mean PM10 concentrations were observed for exposure preceding blood sampling with 3 months to 1 year: for a 10µg/m3 increase in PM10, red blood cell-derived MVs were increased with 77% (95% CI: 14% to 176%) and annexin V-binding derived MVs increased 60% (95% CI: 15% to 123%)
Bollati, 2015 / Air Pollution / Occupational cohort study / 55 / Healthy male steel workers / miFinder RT2 miRNA PCR Arrays / Content / After 3 days of workplace PM exposure, miR-302c and miR-128 expression increased 5.62- and 13.95-foldrespectively (p < 0.001).
Six hour exposure of A549 cells to PM induced a dose-dependent expression of miR-128 in MVs after 6h (p = 0.03), 24h (p = 0.025) and 48h (p = 0.01) of treatment.
Experimental / NA / A549 pulmonary cells / TaqMan® MicroRNA assay / Content
Pavanello, 2016 / Air Pollution / Occupational cohort study / 55 / Healthy male steel workers / miFinder RT2 miRNA PCR Arrays / Content / Increased expression in 17 miRNAs from EV miR-125a-5p-T, miR-126, miR-143, miR-181b, miR-194, miR-196b, miR-200c, miR-21, miR-25, miR-26b, miR-27b,miR-302a, miR-302b, miR-30a, miR-30d, miR-424,miR-9. All were positively associated with PM10 and metal (i.e. Antimony, Arsenic, Copper, Lead, Iron, Nickel, Tin, Zinc and Molybdenum) exposure (p < 0.05, FDR p < 0.1).
Rodosthenous, 2016 / Air Pollution / Cohort Study / 22 / Normative Aging Study / NanoStringnCounter® platform / Content / Ambient PM2.5 exposure during a 6-month window was associated with increased levels in miR-126-3p (Mean ± SD: 0.74 ± 0.21; p = 0.02), miR-19b-3p (0.52 ± 0.15; p = 0.02), miR-93-5p (0.78 ± 0.22; p = 0.02), miR-223-3p (0.74 ± 0.22; p = 0.02), and miR-142-3p (0.81 ± 0.21; p = 0.03). For a 1-year window in ambient PM2.5 exposure a positive association was observed for (0.83 ± 0.23; p = 0.02), miR-150-5p (0.90 ± 0.24; p = 0.02), miR-15a-5p (0.70 ± 0.21; p = 0.02) and miR-191-5p (1.20 ± 0.35; p = 0.02).
Li, 2013 / Smoking / Experimental / NA / THP-1 cells / Flowcytometry (FACSCalibur), Flurogogenic substrate I, Gelatinzymography, Native collagen zymography / Count, Content / Tobacco smoke extract (TSE) exposure for 20 hours caused a quadruplication of MMP14-positive MVs: (mean ± SEM), 1112 ± 231 in control versus 5823 ± 2192 in treated cells. (p < 0.01).
Cordazzo, 2014 / Smoking / Experimental / NA / Normal Human Bronchial Epithelial (NHBE), A549 pulmonary cells / Zymuphen MP-Activity kit, Flowcytometry (FACScantoTM II) / Content, Count / Cigarette smoke extract (CSE) induced a concentration-dependent generation of exosomes after 15 min of exposure in human cells. Statistical significance is reached at 10% CSE (p < 0.05).
Serban, 2016 / Smoking / Case-Control / 25 / Non-smokers, COPD patients / AffymetrixGeneChip® miRNA 3.0 arrays / Content / Cigarette smoke exposure increased exosome levels in plasma of smoking humans (p < 0.001), mice (p < 0.05) as well as supernatants of human cells (p < 0.05). The exosomes levels formiR-126 and miR-125a were increased with 1.11 fold (p < 0.0004) and 1.46 fold (p < 0.02) respectively
Experimental / NA / Mice, THP-1 cells
Fujita, 2015 / Smoking / Experimental / NA / Human Bronchial Epithelial Cells (HBEC) / NanoSight LM10HS, Quant-iT™ Protein Assay (Qubit®2.0 Fluorometer) / Count, Content / Lung fibroblasts were cultured with EVs derived from CSE-induced HBEC. These EVs induced expression of collagen type I compared to non-treated HBEC-derived EVs.
miR-210 showed significant increase in expression (p < 0.05)
Morbarrez, 2014 / Smoking / Crossover / 12 / Healthy Intermittent/sporadic smokers / Flowcytometry (Beckman Gallios) / Origin, Count / A significant increase compared to controls in circulating exosomes of platelet-, endothelial- and leukocyte origin was observed after active smoking of a single cigarette after 1h (control vs smoker; 224 vs 268, p < 0.05), 4h (87 vs 123, p < 0.05), and 24h (176 vs 335, p < 0.05).
Levels of exosomes containing nuclear molecules were increased, of which the majority were positive for CD41 and CD45 (platelet- and leukocyte origin).
Verma, 2016 / Alcohol / Experimental / 29 / Mice / NanoSight NS300, Human cytokine and chemokine antibody-based arrays (ARY05) / Count, Content / Ethanol significantly increased EV release 3.3 fold from hepatocytic cells (p < 0.05) and was associated with activation of caspase-3 (p < 0.05). Alcohol treated hepatocytic cell derived EVs activated THP-1 cells 3.7 fold (p < 0.05). Antibody neutralization demonstrated the necessity of CD40 ligand (CD40L) in EV mediated macrophage activation, causing a significant decrease of THP-1 ability to produce pro-inflammatory cytokines (p < 0.05)
Wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40−/−), experienced less alcohol-induced injury (p < 0.05) and associated macrophage infiltration (p < 0.05)
NA / HepG2 hepatocyte cell line, THP-1 cells
Saha, 2015 / Alcohol / Case-Control / 16 / Alcoholic Hepatitis (AH) patients / Nanosight NS300, SEM (MKII FEI Quanta 200 FEG MKII), Western blotting, Flow Cytometry (BD-LSR II), ELISA / Origin, Content, Count / Ethanol treatment (25-100 nM) resulted in significant dose-dependent increment of the total amount of monocyte-derived EVs (p < 0.05).
Primary human monocytes incubated with ethanol-exposed THP-1 derived EVs had an increased expression of CD14 and CD68 macrophage markers (p < 0.05).
Anti-inflammatory cytokines IL-10 and TGF-β were increased in alcohol EV-treated monocytes compared to controls (p < 0.05). miR-27a expression was increased in EVs derived from ethanol treated monocytes compared to controls (p < 0.05).
AH patients had an increased level of plasma EVs compared to healthy controls (p < 0.05) and had a significantly higher levels of miR-27a in these EVs (p < 0.05).
Experimental / NA / THP-1 cells
Momen-Heravi, 2015 / Alcohol / Case-Control / 24 / Alcoholic Hepatitis patients / NanoSight NS300, Philips CM10 transmission electron microscope, Western blotting, Firefly miRNA multiplex assay, TaqMan®miRNA Assays / Count, Content / Total amount of exosomes was increased in chronic alcohol fed mice sera compared to controls (p < 0.05), as well as in AH patients compared to controls (p < 0.05)
Significant increased levels of miR-122, miR-30a and miR-192 were observed in alcohol fed mice (p < 0.05). The Receiver-Operator Characteristics analyses indicated that miRNA-192 had the highest area under the curve (AUC = 0.96, p < 0.001), miRNA-122 (AUC = 0.92, p < 0.001), and miRNA-30a (AUC = 0.85, p < 0.05) to identify alcohol-induced liver injury in mice.
Both miRNA-192 and miRNA-30a were significantly increased in the circulation of subjects with AH (p < 0.05).
Experimental / 24 / Mice
Hirsova, 2016 / Obesity / Experimental / NA / Primary mouse and human hepatocytes, Huh7 cells / NanoSight NS300, JEOL 1400 electron microscope / Count, Content / Lipotoxicity metabolite lysophospatidyl choline (LPC) treatment caused a 3-fold increase in mouse hepatocyte EV release (p < 0.05) and caused a slight increase in mean size. Human Huh7 cells had an 400-fold increase in EV release (p < 0.05). Similar results were observed for palmitate, a parent compound of LPC.
Campello, 2016 / Obesity / Cohort Study / 20 / Patients with BMI ≥ 40 kg/m3 / Flowcytometry (Cytomics FC500), STA® Procoag PPL assay / Origin, Count / A significant decrease in EVs was observed after weight loss: Annexin V-EV decreased after 3 months (p < 0.001) and 12 months (p < 0.05). Endothelial-derived EVs decreased after 12 months of weight loss (p < 0.001). Platelet-derived, leukocyte derived and tissue factor bearing EVs decreased significantly in the 3rd month (p < 0.0001) and 12th month (p < 0.001).
Heinrich, 2015 / Obesity / Experimental / 20 / Mice / Flowcytometry (FACS cantoTM II) / Count, Content / Obese rats had a 7.5-fold increased amount of MV derived from leucocytes (p = 0.0029) after eating high fat diet (HFD). HFD-fed rats contained 9.4-fold more endothelial cell-derived MV (p = 0.0185). Annexin V platelet-derived MV were 263.7-fold more present in HFD plasma compared to chow-fed rats (p = 0.0039).
VCAM-1 production in HFD-rats increased by 59% compared to chow-fed rats (p = 0.0275).
A 24h treatment, ROS production increased by 30% in HFD versus chow rats (p < 0.0001).
Petrini, 2016 / Obesity / Experimental / NA / Human peripheral blood mononuclear cells (hPBMC) / Flowcytometry (FACS cantoTMII), Zymuphen MP-activity kit / Count / Leptin induced dose-dependent MV production from hPBMC (p < 0.001).
Eguchi, 2016 / Obesity / Experimental / 20 / Mice / high-pressure liquid chromatography (HPLC) coupled with LC-MS/MS, liquid chromatography, TruSeqTM Small RNA kit, flowcytometry (BD LSRII) / Count, Content / Mice were fed a HFD for 2, 4 and 6 weeks, and showed a 3-fold increase in EVs circulation compared to control mice (p < 0.05, p < 0.001 and p < 0.05 respectively). Perilipin A levels were significantly increased in circulating EVs isolated from HFD-fed mice (1.75-fold increase, p < 0.01)).
Obese humans had significantly higher levels of circulating EVs compared to lean controls (p < 0.0001). A calorie restriction period induced a drop in perilipinA abundance in circulating EVs (p < 0.05).
Case-Control / 5 / Humans
Sheller, 2016 / Oxidative stress / Experimental / NA / Primary amnion epithelial cells (AEC) / Flowcytometry (Beckman Coulter), Western blotting / Origin, Content / Heat shock protein 70 (HSP70) and p38 MAPK were present in both CSE treated AEC and controls, but western blot intensity is higher in CSE treated cells. Colocalization of HSP70, p38 MAPK and Histone 3 was significantly higher in CSE treated AEC-derived exosomes compared to controls (p < 0.05).
Atienzar-Aroca, 2016 / Oxidative stress / Experimental / NA / Arising retinal pigment epithelium (ARPE), human umbilical vein endothelial cells (HUVEC) / NanoSight NS300, Flowcytometry (FACScan) Western blotting, qPCR / Count, Content / ARPE-derived exosome release increased after treatment with 80 mM ethanol (p < 0.05). Exosomes contained an increased amount of VEGF-1 and VEGF-2 (p < 0.05 and p < 0.01 respectively), as well as a higher amount of their mRNAs (p < 0.05).
Exosomes derived from ARPE-treated cells caused an increased vascularisation in human umbilical vein endothelial cells (p < 0.05).
Wang, 2015 / Aging / Experimental / NA / lymphoblastoid cell line (LCL) / ChIP Assay, RNA immunoprecipitation / Content / Telomeric repeat-containing RNA (TERRA)were isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. Incubation of TERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of inflammatory cytokines.

Not Available (NA).