Table 3.
Minimal Information for Studies of EVs (MISEV) guidelines related information about EV isolation and characterization in the studies discussed in this review, investigating human-derived EVs. When the effects of human EVs were validated in mice or cell cultures, this is mentioned as ‘additional setup’. For global quantification requirements, consult checkboxes A–E. Checkbox A: Cell number/fluid volume/tissue mass from which EVs were isolated. Checkbox B: Analysis of particle number. Checkbox C: Analysis of protein amount. Checkbox D: Analysis of lipid amount. Checkbox E: Analysis by electron microscopy. For information regarding protein marker detection, consult checkboxes 1–4. Checkbox 1: Transmembrane or Glycosylphosphatidylinositol (GPI)-anchored protein(s) localized in cells at plasma membrane or endosomes. Checkbox 2: Cytosolic protein(s) with membrane-binding or -association capacity. Checkbox 3: Assessment of presence/absence of expected contaminants. Checkbox 4: For small EVs < 200 nm: verifying protein(s) associated with compartments other than plasma membrane or endosomes. Abbreviations: extracellular vesicle (EV), not described (ND), not applicable (NA), time of diagnosis (TOD), post-admission (PA), incubation time (IT), culture medium (CM), lipopolysaccharide (LPS), cecal ligation and puncture (CLP), systemic inflammatory response syndrome (SIRS), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Neisseria meningiditis (N. meningitidis), Streptococcus pyogenes (S. pyogenes), N-formyl-methionyl-leucyl-phenylalanine (fMLP), human umbilical vein endothelial cells (HUVECs), peripheral blood mononuclear cells (PBMCs), pulmonary microvascular endothelial cells (PMECs), brain microvascular endothelial cells (BMECs), cerebral microvascular endothelial cells (CMECs), community-acquired pneumonia (CAP), fecal peritonitis (FP), fecal injection into the peritoneum (FIP), broncho-alveolar lavage (BAL), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), multiple organ dysfunction syndrome (MODS), intravenous (i.v.), intraperitoneal (i.p.), red blood cell (RBC), endothelial cells (ECs), phosphatidylserine (PS), C-reactive protein (CRP), alpha-2-macroglobulin (A2MG), differentially expressed (DE), tissue factor (TF), cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), extracellular superoxide dismutase (SOD), endothelial nitric oxide synthase (eNOS), Nanoparticle Tracking Analysis (NTA), Bicinchoninic Acid Assay (BCA) Bradford assay (BA), mass spectrometry (MS), differential centrifugation (DC), ultracentrifugation (UC), Annexin V (ANX V), flow cytometry (FC), high sensitivity flow cytometry (hsFC).
| Patient Studies | ||||||||
|---|---|---|---|---|---|---|---|---|
| Patient info (disease/controls) |
Biofluid as EV source | Additional setup with EVs | Observations | EV isolation | EV characterization | Ref. | ||
| Global quantification | Protein marker detection | |||||||
| (Severe) sepsis | Healthy controls | Plasma | NA | ↑ Total EV levels ↑ Platelet-, EC-, RBC-, monocyte-, granulocyte- and lymphocyte-EVs in sepsis ↑ PS-EVs in sepsis ↑ Pro-coagulant activity of EVs in sepsis |
Centrifugation Lactadherin-Alexa 488 staining without prior EV isolation |
☑ A (250 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD235a, CD14, CD45, CD66b, CD3, CD19, CD41a, CD31) ☐ 2 ☐ 3 ☐ 4 |
[28] |
| Plasma | Human primary monocytes incubated with EVs | ↑ Total EV plasma levels in sepsis ↑ CRP-EVs in plasma ↑ Platelet-EVs in plasma ↑ IL-8 production by monocytes |
ANX V-PE staining without prior EV isolation or EV-enrichment with centrifugation | ☑ A (500 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD45, CD14, CD235a, CD41) ☐ 2 ☐ 3 ☐ 4 |
[30] | ||
| Plasma Within 24 h TOD |
Vascular ECs treated with EVs (30 min IT, 400 µg/mL protein) | EV-induced, NAD(P)H activity- Dependent, vascular damage and apoptosis. Most EVs of platelet origin (not compared with healthy controls). |
UC ANX V-FITC staining (FC) |
☐ A ☑ B (FC) ☑ C (BA) ☐ D ☐ E |
☑ 1 (CD9, CD63, CD15, CD14, CD61, CD56, CD31, CD42b, CD3) ☐ 2 ☐ 3 ☐ 4 |
[48] | ||
| Plasma Day 1 and 7 PA |
NA | ↑ EV-mRNA for redox genes (D1) EV-miRNA analysis: 30 DE-miRNAs in EVs at D1. 65 DE-miRNAs in EVs at D7. DE-miRNAs in EVs~inflammatory pathways. Partial separation survivors/non-survivors based on 35 EV-associated, DE- miRNAs (~cell cycle regulation). |
UC | ☐ A ☑ B (NTA, nano FC) ☐ C ☐ D ☐ E |
☑ 1 (CD9, CD41) ☑ 2 (FLOT1) ☐ 3 ☐ 4 |
[75] | ||
| Plasma At 0, 24 and 48 h PA |
NA | ↑ EC-EVs ↑ Platelet-EVs (but not significantly) ↑ EC-EVs in the blood of survivors vs. non-survivors. Negative correlation between SOFA score, EC-EV and platelet EV levels. |
ND | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD31, CD42) ☐ 2 ☐ 3 ☐ 4 |
[46] | ||
| Plasma | LPS-stimulated human T-lymphocytes incubated with EVs. EV injection (i.v.) into FIP mice. |
Anti-apoptotic effect of EVs (has-miR-7-5p related). ↓ Bad, Bax, caspase-3 ↑ Bcl2 ↓ Mortality in FIP mice |
DC | ☐ A ☑ B (NTA) ☐ C ☐ D ☑ E |
☑ 1 (CD63) ☑ 2 (HSP70) ☐ 3 ☐ 4 |
[89] | ||
| Plasma Within 24 h PA |
NA | ↑ A2MG, neutrophil derived EVs in sepsis High A2MG-EV levels associated with survival |
Gradient centrifugation and UC | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD66b) ☐ 2 ☐ 3 ☐ 4 |
[79] | ||
| Plasma After admission but before sepsis-related treatment |
Human coronary artery endothelial cells incubated with EVs (24 h IT). | ↑ miR-223 in septic platelet EVs compared to controls EV effects on endothelial cells: ↓ ICAM-1 expression on endothelial cells ↓ PBMC binding to endothelial cells |
DC ANX V-FITC labeling (FC) |
☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD41a) ☐ 2 ☐ 3 ☐ 4 |
[83] | ||
| Plasma | NA | ↑ Pro-coagulant EV activity ↑ PS-EVs |
Centrifugation ANX V-PE labelling (FC) |
☑ A (200 µL) ☑ B (FC) ☐ C ☐ D ☑ E |
☑ 1 (CD14) ☐ 2 ☐ 3 ☐ 4 |
[52] | ||
| Critically ill, non-septic | Plasma Day 1 of diagnosis |
NA | Proteomics on EVs: ↓ Complement, acute phase pathways in sepsis ↑ CRP-EVs in sepsis |
ExoQuick precipitation |
☐ A ☑ B (NTA) ☑ C (MS, BA) ☐ D ☐ E |
☐ 1 ☑ 2 (actin) ☑ 3 (albumin) ☐ 4 |
[73] | |
| Plasma Day 1 |
Lymphocytes incubated with EVs |
Pro-apoptotic effect of EVs (caspase-1 dependent). Caspase-1 activity in EVs higher in plasma EVs from sepsis patients. |
DC | ☐ A ☑ B (FC) ☐ C ☐ D ☑ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[90] | ||
| None | Plasma Six timepoints during disease progression |
NA | Negative correlation between EV-SPTLC3 protein and disease progression (~body temperature, CRP levels). | DC + 17% Optiprep | ☑ A (300 µL) ☑ B (NTA) ☑ C (MS) ☐ D ☑ E |
☑ 1 (CD63) ☐ 2 ☐ 3 ☐ 4 |
[119] | |
| Plasma | EV incubation with tissue-engineered vascular media (1417–48190 EVs/μL, 24 h IT) | Restoration of vascular hypo-reactivity (IL-10 mediated) | DC | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD41, CD45, CD235a, CD146, CD11b, CD66b, CD62L, CD62P) ☐ 2 ☐ 3 ☐ 4 |
[124] | ||
| SIRS patients and healthy controls |
Plasma | NA | ↑ TF+/CD13+ monocyte-derived EVs. High levels of TF+/CD13+ EVs correlated with higher APACHE II score and DIC scores. |
ANX V labeling of plasma without prior EV isolation | ☑ A (50 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD13, CD142) ☐ 2 ☐ 3 ☐ 4 |
[110] | |
| Plasma | NA | ↑TF-EVs from ECs High amount of TF-EVs correlated with high DIC score. Negative correlation between the ratio of thrombomodulin (TM)- and EC-derived TF- EVs with DIC scores. |
ND | ☑ A (50 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD146, CD142, CD141, CD201) ☐ 2 ☐ 3 ☐ 4 |
[45] | ||
| Blood | NA | ↑ Platelet-EVs | ND | ☑ A (50 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD42a, CD62P) ☐ 2 ☐ 3 ☐ 4 |
[40] | ||
| Sepsis + MODS | Healthy controls | Plasma | NA | ↑ Granulocyte-EVs ↓ EC-EVs, platelet-EVs Thrombin generation negatively correlated with EV numbers. |
Centrifugation ANX V-PE labeling |
☑ A (250 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD61, CD235a, CD62E, CD144, CD66b) ☐ 2 ☐ 3 ☐ 4 |
[41] |
|
Sepsis +
CAP or FP |
Plasma Day 1,3 and 5 PA |
NA | ↑ Neutrophil-, monocyte-, lymphocyte- and EC-EVs in CAP vs. FP and healthy controls. No difference in RBC- and platelet-EVs between CAP/FP/healthy controls. High A2MG-EV levels are associated with survival of CAP patients |
DC ANX V-Pacific Blue labeling |
☑ A (5 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD66b, CD14, CD235, CD41, CD3, CD51, CD146) ☐ 2 ☐ 3 ☐ 4 |
[42] | |
|
Sepsis +
CAP |
Plasma Day 1 |
NA | ↑A2MG levels in neutrophil-derived EVs | DC | ☐ A ☑ B (FC) ☑ C (MS) ☐ D ☐ E |
☑ 1 (CD66b, CD45, CD62P; CD14, CD41, CD54) ☑ 2 (ANXA1, HSP71, Actin) ☐ 3 ☐ 4 |
[118] | |
| ARDS patients | None | Plasma BAL fluid Day 1 and 3 of ARDS |
NA | Increased levels of leukocyte-EVs are associated with better survival. | Plasma: ND BAL: DC |
☑ A (30 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD31, CD41, CD45, CD11b, CD66b) ☐ 2 ☐ 3 ☐ 4 |
[50] |
| Septic shock | Healthy controls | Plasma | NA | No significant difference in platelet-EVs between septic shock and healthy controls. Lower amount of platelet EVs in patients with DIC score > 5 compared to DIC score < 5. ↑ CD144 and CD62E+ EC-EVs in septic shock (in low range of detection limit). ↑ CD41+ EVs, CD31+/CD41- EVs plasma of septic shock patients who died within 48 h after inclusion vs. survivors. |
ANX V-FITC staining of plasma without prior EV isolation | ☐ A ☑ B (hsFC) ☐ C ☐ D ☐ E |
☑ 1 (CD144, CD42b, CD62E, CD106, CD41, CD31, CD45, CD66b, CD20, CD14, CD3) ☐ 2 ☐ 3 ☐ 4 |
[44] |
| Plasma 10 ± 4 h after enrollment |
EV injection (i.v.) in LPS-treated mice (40 mg/kg, i.p.) | ↑ Total EV levels in septic shock ↑ L- and P-selectin in EVs ↑ EC-EVs and platelet-EVs ↓ Leukocyte-EVs No difference in RBC-, monocyte- and granulocyte-EVs. Counter-act hypo-reactivity in the aorta (↑ thromboxane A2) |
DC | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD41, CD45, CD235a, CD146, CD11b, CD66b, CD62L, CD62P) ☑ 2 (actin) ☐ 3 ☐ 4 |
[29] | ||
| Plasma | Rabbit-derived heart preparations incubated with EVs (0.5 to 1× EV amount in plasma) | EV-induced nitric oxide production. Induction of myocardial dysfunction. |
UC | ☐ A ☐ B ☑ C (BA) ☐ D ☑ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[125] | ||
| None | Plasma Day 1, 3 and 7 |
NA | ↑ PS-EVs Combination of prothrombin time, EC-derived CD105+ EVs and platelet count at D1 could predict DIC absence. |
ANX V labeling of plasma without prior EV isolation | ☐ A ☐ B ☐ C ☐ D ☐ E |
☑ 1 (CD31, CD105, CD11a) ☐ 2 ☐ 3 ☐ 4 |
[95] | |
| Septic shock + DIC | Septic shock without DIC | Plasma Day 1, 3, 5, 7 |
NA | Total EV amount in same range for DIC and non-DIC patients. Specific EV source pattern in DIC patients. ↓ Platelet-derived EVs in DIC patients compared to non-DIC patients ↑ EC-EVs and leukocyte EVs in DIC (D1) ↑ Leukocyte- EVs in DIC at D7 |
ND | ☐ A ☐ B ☐ C ☐ D ☐ E |
☑ 1 (CD11a, GPIb, CD31, CD62E, CD105) ☐ 2 ☐ 3 ☐ 4 |
[51] |
|
(Severe) sepsis
septic shock |
Healthy controls | Serum | NA | No difference in EV levels between sepsis/septic shock patients and healthy donors. miR125-5p exclusively upregulated in serum EVs from sepsis patients (~survival prediction). |
miRCURY Exosome Isolation kit | ☑ A (3 mL) ☑ B (NTA) ☐ C ☐ D ☑ E |
☑ 1 (CD81) ☑ 2 (TSG101, syntenin-1) ☐ 3 ☐ 4 |
[33] |
| Plasma 10 ± 4 h after enrollment |
EVs injected (i.v.) in Swiss mice. | Organ damage propagation via oxidative stress and inflammation: Heart: ↑ COX1, COX2, SOD, eNOS. Lungs: COX-2, NFκB levels affected. Liver: ↓ eNOS, manganese SOD. |
ND | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD62L, CD62P) ☐ 2 ☐ 3 ☐ 4 |
[78] | ||
| Plasma | NA | No difference in EC-EV levels | No EV isolation | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD31, CD42b) ☐ 2 ☐ 3 ☐ 4 |
[47] | ||
| Plasma Within 48 h after enrollment |
NA | EV levels: septic shock > sepsis without shock > healthy controls. High EV level group associated with development of septic shock, the need for mechanical ventilation or vasopressor support, disease severity (SAPS 3, APACHE II and SOFA scores), and 28-day mortality. |
ExoQuick precipitation |
☑ A (250 µL) ☐ B ☑ C (BCA) ☐ D ☑ E |
☑ 1 (CD9, CD63) ☐ 2 ☐ 3 ☐ 4 |
[31] | ||
| Critically ill non-septic and healthy controls |
Plasma Day 1, 3 and 5 from TOD |
NA | ↑ EV levels in sepsis/septic shock vs. healthy controls. ↑ EV levels in septic shock patients vs. sepsis patients. No difference in EV levels between sepsis patients and critically ill, non-septic patients. ↑ De novo methylation regulators DNMT3A and DNMT3B mRNAs in EVs in the septic shock cohort vs. critically ill, non-septic control and sepsis cohorts. |
DC | ☑ A (1 mL) ☑ B (NTA) ☐ C ☐ D ☐ E |
☑ 1 (CD63, CD81, EPCAM) ☑ 2 (FLOT1, TSG101, ANXA5) ☐ 3 ☑ 4 (GM130) |
[27] | |
|
Sepsis
vs. Severe sepsis vs. Septic shock |
Plasma Daily for 2 weeks |
NA | Higher amount of PS-positive EVs correlated with a lower risk for mortality and multiple organ failure. High amounts of TF correlated with increased risk for high disease severity (SAPS II score > 60). | ND | ☐ A ☐ B ☐ C ☐ D ☐ E |
☑ 1 (CD142) ☐ 2 ☐ 3 ☐ 4 |
[116] | |
|
Human endotoxemia
(2 ng/kg) |
None | Plasma 0, 3, 6 and 24 h post-LPS |
NA | ↑ Total and platelet-EVs 6 h post-LPS ↑ PS-EVs ↑ EV-associated TF activity 6 h post-LPS |
ANX V-FITC staining of plasma without prior EV isolation | ☑ A (30 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD31, CD41a, CD14, CD235a) ☐ 2 ☐ 3 ☐ 4 |
[32] |
| Healthy controls | Plasma 0, 1, 2, 3, 4, 8, and 24 h post-LPS |
NA | ↑TF activity of EVs post-LPS | UC and ANX V-Cy5 labelling | ☐ A ☑ B (FC) ☐ C ☐ D ☑ E |
☑ 1 (CD14, CD144) ☐ 2 ☐ 3 ☐ 4 |
[104] | |
| Healthy donors | Plasma | NA | Most EVs are platelet-derived. Low amount of TF-bearing EVs detected (median 47 × 106 EVs/L). EV-dependent, low grade thrombin generation was TF-independent. |
Centrifugation ANX V-PE staining |
☑ A (250 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD14, CD61, CD62E, CD66e, CD235a) ☐ 2 ☐ 3 ☐ 4 |
[36] | |
| Plasma | NA | Most EVs are platelet-derived. | Lactadherin-FITC staining without prior EV isolation | ☐ A ☑ B (FC, ImageStream) ☐ C ☐ D ☐ E |
☑ 1 (CD14, CD41, CD235a, CD45) ☐ 2 ☐ 3 ☐ 4 |
[37] | ||
| Plasma | NA | Most EVs are platelet-derived. | Lactadherin-FITC staining of plasma without prior EV isolation | ☐ A ☑ B (FC, ImageStream) ☐ C ☐ D ☐ E |
☑ 1 (CD41, CD235a) ☐ 2 ☐ 3 ☐ 4 |
[38] | ||
| Plasma | NA | Generally more inside EVs: IL-2, IL-4, IL-10, IL-12, IL-15, IL-16, IL-18, IL-21, IL-22, IL-33, Eotaxin, IP-10, ITAC, M-CSF, MIG, MIP-3α, TGF-β, and TNF. Generally more on EV surface: IL-8, IL-17, and GRO-α. Specific for IL-6: Selectively bound to the EV surface when released from tissues, but mostly present inside EVs derived from body fluids or cultured immune cells. Cellular activation influences cytokine secretion pattern of monocytes (stimulus-dependent). Monocytes + LPS IL-1α, IL-1β, IL-10, IL-18, IL-21, IL-22, GM-CSF, Gro-α, and TNF: ↓ EV-associated secretion, MCP-1: ↑ EV-associated secretion Shift EV surface-EV encapsulation: for IL-1β, IL-18, GRO-α, IP-10, M-CSF, MCP-1 and MIP-1α. |
ExoQuick (TC) precipitation | ☑ A (250 µL) ☑ B (NTA) ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[58] | ||
| Amniotic fluid | NA | ND | ☐ A ☑ B (NTA) ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[58] | |||
| Explant CM: Placental, tonsillar and cervix tissue. CM cell culture: T-cells and monocytes. |
NA | ExoQuick (TC) precipitation |
☑ A (500 µL) ☑ B (NTA) ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[58] | |||
| Plasma/platelet concentrates |
NA | Total EV amount: 2× 105 EVs/µL PS-EVs (10%) were mainly platelet-derived (88.4%). PS-EVs: PS- dependent thrombin generation. |
DC or Total Exosome Isolation kit ANX V-FITC labelling (FS) Lactadherin-FITC-labelling (ImageStream) |
☐ A ☑ B (NTA, FC, ImageStream) ☑ C (ND) ☐ D ☑ E |
☑ 1 (CD14, CD41, CD235a, CD45) ☐ 2 ☐ 3 ☐ 4 |
[49] | ||
| Bacteremic S. aureus | Healthy controls | Serum | NA | ↑ Granulocyte-EVs | Filtering and sedimentation | ☐ A ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD11b, CD177) ☐ 2 ☐ 3 ☐ 4 |
[43] |
| Meningococcal sepsis | Healthy controls | Plasma | NA | ↑ Platelet-EVs ↑ Granulocyte-EVs ↑ PS- EVs, mainly derived from platelets. ↑ TF-EVs (85% monocyte-derived), with higher TF- and pro-coagulant activity (especially in DIC patients) |
Centrifugation ANX V-PE staining |
☑ A (250 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD4, CD8, CD14, CD20, CD61, CD62E, CD235a, CD66b) ☐ 2 ☐ 3 ☐ 4 |
[39] |
| Meningococcal septic shock | Meningococcal meningitis | Plasma | NA | More efficient TF-dependent thrombin generation and clot formation by TF-EVs in meningococcal septic shock vs. patients with meningitis. EV-TF activity was associated with plasma LPS levels. |
DC | ☑ A (300 µL) ☐ B ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[107] |
| Cardiac surgery patients | Healthy controls | Plasma | NA | No difference in EV levels. ↑ TF+ EVs in cardiac surgery patients ↑ Pro-coagulant activity of TF-EVs from cardiac surgery patients. Healthy controls: 5% of EVs was TF+, no pro-coagulant activity in vitro. |
Centrifugation and ANX V-APC labelling | ☑ A (250 µL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD14, CD66e, CD142, CD61, CD235a) ☐ 2 ☐ 3 ☐ 4 |
[101] |
| Burn patients | Healthy controls |
Plasma (0–120 h AD) |
NA | ↑ HMGB1-EVs in burn patients | DC | ☐ A ☑ B (FC) ☑ C (BCA) ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[62] |
| In vitro studies | ||||||||
| Cells | Stimulus | Additional setup with EVs | Observations | EV isolation | EV characterization | Ref. | ||
| Global quantification | Protein marker detection | |||||||
| THP-1 monocytes | LPS from E. coli 100 µg/mL, 20 h |
NA | Release of CD31+/CD41- EVs. 26.8% expressed at least one of the analyzed leukocyte markers (CD45, CD14, CD66b, CD20 or CD3). |
ANX V-FITC staining of CM without prior EV isolation | ☑ A (2 × 106 cells/mL) ☑ B (hsFC) ☐ C ☐ D ☐ E |
☑ 1 (CD41, CD31, CD45, CD66b, CD20, CD14, CD3) ☑ 2 ☐ 3 ☐ 4 |
[44] | |
| LPS 5 µg/mL, 4 h |
NA | ↑ TF expression on monocyte-derived EVs from LPS-stimulated cultures vs. EVs from unstimulated monocyte cultures. TF blockage: ↓ thrombin generation by EVs from LPS-stimulated monocytes. |
DC | ☑ A (1 × 106 cells/mL) ☐ B ☑ C (ND) ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[49] | ||
| LPS from E. coli (6 h and 24 h IT) Hyperthermia (37 °C vs. 39.5 °C, 6 h and 24 h IT) |
NA | LPS + hyperthermia: EV-independent release of inducible HSP70. EV dependent release of constitutive HSP70. |
DC | ☐ A ☐ B ☐ C ☐ D ☑ E |
☑ 1 (CD63) ☑ 2 (HSP70) ☐ 3 ☐ 4 |
[61] | ||
|
PBMCs
(healthy volunteers) |
S. pyogenes | S. pyogenes-infected Balb/c mice treated with EVs from stimulated PBMCs (50 to 150 EVs/mL) | ↑ Fibrinogen-binding integrins in EVs Anti-bacterial effect of EVs: bacteria trapping into fibrin networks. ↓ Bacterial loads in the blood, spleen and liver in S. pyogenes-infected mice |
DC | ☑ A (2.6 × 106 PBMCs, 50–150 EVs/mL) ☐ B ☑ C (MS) ☐ D ☑ E |
☑ 1 (CD14, CD45, CD18) ☑ 2 (ANXA1, ANXA2, ANXA5, ANXA6) ☐ 3 ☐ 4 |
[88] | |
| M proteins from S. pyogenes (1 mg mL, 24 h IT) LPS (100 ng mL, 24 h IT) Lipoteichoic acid (1 mg mL, 24 h IT) |
NA | Higher abundance of PS and TF on EVs from stimulated PBMCs. Both intrinsic and extrinsic coagulation pathways are involved in EV-triggered clotting. |
DC ANX V-PE labelling (FC) |
☑ A (900 µL) ☑ B (FC) ☐ C ☐ D ☑ E |
☑ 1 (CD14) ☐ 2 ☐ 3 ☐ 4 |
[52] | ||
| LPS from E. coli 1 µg/mL | Vascular smooth muscle cells incubated with EVs | Monocyte-derived, caspase-1-carrying EVs can induce apoptosis in smooth muscle cells. | DC | ☑ A (10 × 106 cells/mL) ☑ B (FC) ☐ C ☐ D ☑ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[123] | ||
| LPS 6 ng/mL, 12 h |
Naïve human monocytes incubated with EVs from LPS-treated monocytes (12 h IT), followed by LPS stimulation (6 ng/mL, 4 h IT) | Proteomics on EVs: ↓ Complement and acute phase pathways post-LPS In vitro validation: ↓ TNF expression by monocytes |
ExoQuick precipitation |
☐ A ☑ B (NTA) ☑ C (MS, BA) ☐ D ☐ E |
☐ 1 ☑ 2 (actin) ☑ 3 (albumin) ☐ 4 |
[73] | ||
| N. meningitidis (4 h) | NA | ↑ TF activity in monocytes and EVs exposed to LPS from N. meningitidis | DC ANX V-FITC labelling |
☑ A (106 cells) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD14, CD142) ☐ 2 ☐ 3 ☐ 4 |
[113] | ||
| Primary neutrophils | Various stimulants (e.g., LPS, S. aureus, PMA, TNF) | NA | Highest amount of EV production by S. aureus. Anti-bacterial effect of EVs due to EV-bacteria aggregation. Interference with actin polymerization, glucose metabolisms and β2-integrins, impaired anti-bacterial effect of EVs. |
Filtering and sedimentation ANX V-FITC labelling |
☑ A (4.5 × 106 cells) ☑ B (FC) ☑ C (BA) ☑ D (MS) ☑ E |
☑ 1 (CD11b, CD18) ☑ 2 (Actin) ☐ 3 ☐ 4 |
[43] | |
| S. aureus, E. coli or LPS (1 µg/mL) | Stimulation of THP-1 monocytes with EVs (108 particles) or LPS (100 ng/mL) | “Trails” = neutrophil-derived EVs produced during migration towards inflamed foci. Anti-bacterial effect: reactive oxygen species (ROS)- and granule-dependent. Monocyte attraction (MCP-1) Induction of macrophage polarization to pro-inflammatory phenotype. Pro-inflammatory miRNAs profile (miR-1260, miR-1285, miR-4454, and miR-7975) in trails vs. anti-inflammatory miRNAs (miR-126, miR-150, and miR-451a) in neutrophil-derived released upon arrival at the inflamed foci. |
Filtering and DC | ☑ A (2 × 109 cells) ☑ B (NTA) ☐ C ☐ D ☑ E |
☑ 1 (CD81, CD63, CD9, CD66b, CD35, CD11b, CD39, CD29, CD18) ☑ 2 (ANXA1, FLOT-1, HSP70) ☐ 3 ☐ 4 |
[74] | ||
| +/− pre-incubation (20 min) with HUVEC monolayer. fMLP (1 µM, 20 min) |
A2MG- EV injection (105 EVs, i.v., 1 h post-CLP) in CLP mice (2 punctures with 20 G). A2MG-EVs (5 × 104/0.6 cm2 channel, 4 h IT) incubated with TNF-stimulated HUVECs (10 ng/mL TNF, 4 h IT) |
Beneficial effects of A2MG-EVs in CLP mice ↑ Survival ↓ Pro-inflammatory cytokine levels in plasma ↓ Bacterial load ↑ Bacterial phagocytosis in peritoneal exudates ↓ Neutrophil infiltration lungs ↓ Hypothermia A2MG transfer to plasma membrane of ECs. ↑ Neutrophil adhesion to HUVECs |
DC | ☑ A (2 × 107 cells) ☐ B ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[79] | ||
| fMLP (1 µM, 20 min) | HUVECs incubated with EVs (8 × 105, 6 h) | Anti-inflammatory effects (↓STAT1, NFKBIZ, CCL8, or CXCL6 in HUVECs) | DC | ☑ A (2 × 107 cells/mL) ☑ B (FC) ☑ C (MS) ☐ D ☐ E |
☑ 1 (CD66b) ☑ 2 (ANXA1, HSP71, actin) ☐ 3 ☐ 4 |
[118] | ||
| Ca2+ ionophore (2 µM, A23187, 20 min) | HUVECs incubated with EVs (30 min IT) | MPO-EVs induce EC damage. | DC PKH6 and ANX V-PE labelling |
☐ A ☑ B (FC) ☑ C (BCA) ☐ D ☑ E |
☑ 1 (CD66b, CD62L) ☐ 2 ☐ 3 ☐ 4 |
[121] | ||
|
Blood monocytes
(healthy donors) |
LPS from E. coli, 10 ng/mL, 4 h | NA | TF-pro-coagulant activity assay Constitutive generation of TF-EV generation barely detectable in CM at baseline. ↑ TF-EV generation post-LPS. |
ND | ☐ A ☐ B ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[104] | |
| Whole blood | N. meningitidis (106/mL, WT and LPS-deficient). LPS from N. meningitidis and E. coli (4 h IT). |
NA | In vitro whole blood model to study TF activity: ↑ TF activity in EVs from in whole blood after exposed to WT N. meningitidis vs. LPS-deficient N. meningitidis. ↓ TF activity of EVs from whole blood after exposure to LPS from N. meningitidis or E. coli vs. EVs released after exposure to N. meningitidis. |
DC | ☑ A (250 µL) ☐ B ☐ C ☐ D ☐ E |
☑ 1 ☐ 2 ☐ 3 ☐ 4 |
[113] | |
|
Platelets
(healthy donors) |
Platelet labeling with CellTracker Orange CMTMR. Platelet activation with thrombin (0.1 U/mL, 15–60 min IT). |
HUVECs incubated with EVs (ratio 1:100 HUVEC:EVs, 48 h IT) | EV internalization by HUVECs. miR-223 in platelet EVs is transferred to HUVECs and can regulate the expression of endothelial genes (FBXW7 and EFNA1). |
Filtering and DC | ☑ A (108 platelets/mL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD41a) ☐ 2 ☐ 3 ☐ 4 |
[82] | |
| Platelet labeling with CellTracker Orange CMTMR or CellTracker Red CMPTX dye. Platelet activation with thrombin (0.1 U/mL, 60 min IT). |
PBMCs incubated with EVs (ratio 1:100, 6 h IT). | EV internalization by macrophages. EV effects on macrophages: ↑ Phagocytic activity ↓ Cytokine and chemokine release (CCL4, TNF, M-CSF) (mi)RNA expression analysis in macrophages exposed to platelet EVs. 66 miRNAs and 653 additional RNAs differentially expressed. Upregulation of 34 miRNAs, concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines. |
Filtering and DC | ☑ A (108 platelets/mL) ☑ B (FC) ☐ C ☐ D ☐ E |
☑ 1 (CD41a) ☐ 2 ☐ 3 ☐ 4 |
[87] | ||
| HUVECs | Transfection with Ad-HSPA12B-GFP or Ad-GFP. +/− LPS stimulation (4 h IT) |
LPS-treated RAW264.7 macrophages and BMDMs (24 h LPS, 1 µg/mL LPS) incubated with EVs (1 h IT) | Endothelial HSPA12B-EVs Uptake by macrophages. EV effects in LPS-stimulated macrophages: ↑ IL-10 production ↓ TNF, IL-1 production ↓ NFκB activation |
PEG6000 | ☐ A ☐ B ☐ C ☐ D ☐ E |
☑ 1 (CD9) ☑ 2 (HSPA12B) ☐ 3 ☐ 4 |
[63] | |
| TNF stimulation (10 ng/mL, 4 h) | NA | TF- pro-coagulant activity assay Constitutive generation of TF-EV generation barely detectable in CM at baseline. ↑ TF-EV generation post-TNF. |
ND | ☐ A ☐ B ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[104] | ||
| EPCs | Isolated from cord blood of healthy pregnant women. | Intratracheal administration of EVs to LPS-induced ALI mice (25 µg LPS) | Beneficial effects on lung damage in acute lung injury mice(partly miRNA-126 related). ↓ Cell number, protein concentration and cytokine/chemokine concentration in BAL fluid. ↓ Edema, myeloperoxidase activity and organ damage score in lungs. |
Total Exosome Isolation kit | ☐ A ☑ B (NTA) ☑ C (Biorad DC protein assay) ☐ D ☐ E |
☑ 1 (CD81, CD9, CD63) ☐ 2 ☐ 3 ☐ 4 |
[117] | |
| PMECs | TNF (100 ng/mL, 24 h) | Pulmonary microvascular ECs incubated with EVs (10 µg/mL, 24 h IT) | EC-derived EVs can induce inflammatory pathways in parent ECs itself. ↑ IP-10 expression, NFκB translocation |
DC | ☐ A ☐ B ☐ C ☐ D ☐ E |
☐ 1 ☐ 2 ☐ 3 ☐ 4 |
[122] | |
| Human mast cells (HMC-1) | NA | NA | Protein topology analysis of EVs: many cytosolic proteins are situated on the EV surface. | DC and discontinuous iodixanol density gradient | ☑ A (5 × 105 cells/mL) ☑ B (NTA) ☑ C (MS, BCA) ☐ D ☑ E |
☑ 1 (CD81, CD63) ☑ 2 (TSG101, FLOT-1, GAPDH) ☐ 3 ☑ 4 (Histone H1) |
[57] | |
| BMECs | Mechanical injury (mechanical strain-induced injury model) |
NA | Time-dependent increase in EV-associated occludin, CD31 and ICAM-1 following mechanical injury. | ExoQuick TC precipitation |
☐ A ☐ B ☐ C ☐ D ☐ E |
☑ 1 (CD31) ☐ 2 ☐ 3 ☐ 4 |
[132] | |
| CMECs | TNF (10 ng/mL, 24 h IT) | NA | ↑ Adhesion molecules in EVs post-TNF stimulation (ICAM-1 and VCAM-1). | DC | ☑ A (13–18 × 106 cells) ☑ B (NTA) ☑ C (BA, MS) ☐ D ☑ E |
☑ 1 (CD9, CD81) ☑ 2 (ALIX) ☐ 3 ☑ 4 (HSP90B1) |
[133] | |