APC-released EVs promote anti-inflammation and endothelial barrier protection. Primary cultures of human umbilical vein endothelial cells (HUVEC, passages between 3 to 6) were grown to confluency in endothelial basal growth medium (EBM-2; Lonza) in 6-well culture plates. When cells reached confluency, they were washed once with a serum-free EBM-2 medium (lacks growth factors and serum) before they were subjected to experimental treatments. (A) APC increases EV release from endothelial cells. HUVECs grown in 6-well culture plates were treated with a control vehicle (Control) or APC (100 nM) in 1 ml of serum-free EBM-2 medium for 8 hours. EVs were isolated from the culture supernatant media, as described previously.⁴ Briefly, the supernatant media was centrifugated at 2500g for 10 minutes at 4°C to sediment cells and cell debris. The pellet was suspended in 1 mL Hanks' balanced salt solution (HBSS) and centrifugated at 21 000g for 60 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of HBSS and the EV suspension was recentrifugated for 60 minutes at 4°C. The aforementioned step was repeated once more, and the final EV pellet was resuspended in 1 mL HBSS. EV count was quantified by nanoparticle tracking analysis (NTA) by Nano Sight NS300 (Malvern Panalytical). (B) Protein content–based EV characterization. Based on MISEV 2018 guidelines,⁸ EV nature and the degree of purity of EV preparation was analyzed by the presence of transmembrane proteins associated to plasma membrane (CD63, CD31, and EPCR), cytosolic proteins recovered in EVs (HSP70 and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), potential components of non-EV coisolated proteins (albumin), or soluble proteins associated with intracellular compartments other than plasma membrane/endosomes (calnexin) by immunoblot analysis. EVs isolated from an equal volume of conditioned media of endothelial cells treated with a control vehicle (control) or APC were used for the immunoblot analysis. Cell extracts of the same treatments were used as controls. (C) Levels of APC associated with the EVs. APC levels associated with control vehicle- or APC-released EVs were measured by chromogenic assay. (D-E) APC-released EVs confer anti-inflammatory phenotype ro recipient cells. THP-1 cells were incubated with a control vehicle (Control) or EVs derived from HUVECs treated with a control vehicle (Con EVs) or APC (APC EVs) for 4 hours in equal numbers (2 × 10⁸; cells-to-EV ratio, 1:100). After the cells were washed to remove the free EVs, they were challenged with LPS (200 ng/mL). After 12 hours, the levels of proinflammatory cytokines, tumor necrosis factor α (TNF-α) (D) and interleukin-6 (IL-6) (E), in the supernatant medium were determined by enzyme-linked immunosorbent assay. (F) APC-released EVs protect aganist endothelial barrier disruption. HUVECs grown to confluence in transwells were incubated with a control vehicle (Control) or EVs (2 × 10⁸) released from HUVECs treated with control vehicle (Con EVs) or APC (APC EVs) for 4 hours. After 4 hours, the monolayer was washed twice and challenged with LPS (200 ng/mL). Barrier permeability was measured 12 hours after the addition of LPS by endothelial barrier permeability in vitro assay using Evans blue–labeled bovine serum albumin. The barrier permeability (optical denisty readings) observed in cells treated with LPS that were not exposed to EVs were taken as 100%. (G-I) Cytoprotective effects of APC-EVs are independent of EVs’ bound APC. THP-1 cells or naïve HUVECs were preincubated with EPCR-blocking antibody (EPCR B-Ab; JRK1494; 100 μg/mL) or nonblocking control antibody (EPCR NB-Ab; JRK1500; 100 μg/mL) for 1 hour before the addition of control EVs or APC-EVs. In a subset, EVs were preincubated with anti-APC polyclonal antibodies (APC B-Ab; 100 μg/mL) or control immunoglobulin G (IgG; 100 μg/mL) for 1 hour followed by the fusion with recipient THP-1 cells and naïve HUVECs. EVs-fused recipient cells were challenged with LPS, and the release of TNF-α (G) and IL-6 (H) from THP-1 was measured by enzyme-linked immunosorbent assay, and barrier permeability (I) was determined in HUVECs as described earlier. (J-M) In vivo cytoprotective effects of APC-released EVs. EVs were isolated from the supernatant medium of bEND.3 cells that were treated with a control vehicle or APC and an equal number of EVs (2 × 10⁸) were administered to wild-type mice (C57BL/6J, between 8 and 12 weeks old, male and female equally distributed) via the tail vein. In additional experimental groups, mice were administered with APC-EVs that were preincubated with polyclonal antibodies against APC (APC B-Ab; 100 μg/mL) or control IgG (100 μg/mL) for 1 hour. Four hours later, mice were given an intraperitoneal injection of LPS (5 mg/kg). Twelve hours after the administration of LPS, blood was obtained from the mice, and the levels of TNF-α (J) and IL-6 (K) in the plasma were measured. In a subset of the same group of mice, vascular leakage into the heart (L) and liver (M) was evaluated, as described in our earlier study.⁵ All aforementioned in vitro experiments were repeated independently at least 3 times. Six to 8 mice per group were used for in vivo studies. Data are shown as mean ± standard error of the mean. Statistical significance among multiple groups was analyzed by a 1-way analysis of variance followed by the Tukey post hoc test. Statistical significance between the 2 groups was calculated by using the Mann-Whitney U test; ∗P < .05; ∗∗∗P < .001; and ∗∗∗∗P < .0001. ns, not statistically significant.