Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles

Abstract

Aims Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell–cell signalling and are thus a potential mechanism for communicating with remote tissues. Methods and results Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Conclusions Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Comparative and Integrated Analysis of Plasma Extracellular Vesicles Isolations Methods in Healthy Volunteers and Patients Following Myocardial Infarction

“…The tubes were filled with a 16 G hypodermic needles (Microlance, VWR, Pennsylvania, United States) fitted to a 10 mL syringe (VWR, Pennsylvania, United States) and after plasma was injected into the tube, 13 mL of phosphate buffered saline (PBS, Thermo Fischer Scientific, Massachusetts, United States) was added. The tubes were sealed using a soldering iron (Zacro, 60 W) and centrifuged using an Optima MAX-XP ultracentrifuge (Beckman Coulter, California, United States) at 120,000 x g for 120 minutes at 4 °C with a MLA55 fixed-angle rotor (Beckman Coulter, California, United States) [5, 6]. The pelleted plasma EV were resuspended in 100 µL PBS or RIPA buffer (Thermo Fischer Scientific, Massachusetts, United States) for subsequent analysis.…”

“…Plasma EV size distribution and concentration were determined by Nanoparticle Tracking Analysis (NTA) using a Zetaview device (Particle Metrix, Inning am Ammersee, Germany) as previously described [5, 6]. Prior to injection into the sample chamber, samples were diluted in PBS.…”

“…Isolated plasma EV were analysed by a targeted EV-Array, which has been described previously [6, 34]. In short, a protein microarray plate was generated with the following antibodies: CD146 (P1H12), Flotillin-1, TSG101 (Abnova, Taiwan), CD9, CD81 (Ancell corporation, Minnesota, United States); CD16 (3G8, BD Biosciences, California, United States); Alix (3A9), VEGFR2 (7D4-6; Biolegend, California, United States); CD63 (Bio-Rad, California, United States); ICAM-1 (R6.5, eBioscience, California, United States); Endoglin (LSbio, Washington, United States); Tissue factor (323,514), VCAM-1 (HAE-2Z), Thrombomodulin (501733), CD31 (AF806, R&D Systems, Minnesota, United States), VE-Cadherin (AF938, R&D Systems, Minnesota, United States).…”

“…Isolated plasma EV or controls were lysed using RIPA buffer with protease and phosphatase inhibitors PhosSTOP (Roche, Basel, Switzerland) and cOmplete (Roche, Basel, Switzerland) and were needle sonicated by a SonoPuls HD2070 (Bandelin, Berlin, Germany) at 40% power for 10 seconds as previously described [5, 6]. Following sonication, samples were incubated at 95 °C for 5 minutes to reduce.…”

“…We then sought to determine how plasma EV isolation methods influence EV characteristics in a set of clinically well characterised individuals. Acute myocardial infarction (MI) is an important pathology; it is also an example of sterile inflammation where plasma EV number and composition are acutely altered [5, 6]. Plasma EV from MI patients at two different time points were isolated using the five different EV isolation methods ( Figure 1 ).…”