Ischaemia alters the effects of cardiomyocyte‐derived extracellular vesicles on macrophage activation

Abstract Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell‐cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte‐derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro‐inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL‐1β and IL‐6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL‐1β, IL‐6, IL‐10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum‐circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte‐derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post‐infarction remodelling.


EV isolation from human serum samples
Venous blood samples were collected into a non-heparinized tube (BD Vacutainer SST II Plus plastic serum tube, BD Biosciences) within 12 hours after admission to the Coronary Intensive Care Unit, in the case of AMI patients, or within 20 minutes after coronary angiography in the controls. Blood was allowed to clot at room temperature (RT) for approximately 30 min, after which serum was retrieved in the supernatant by centrifugation at 1.000 x g for 15 min at RT.
Serum samples were diluted in PBS (1:2), centrifuged at 2.000 x g for 30 min, followed by 45 min at 12.500 x g. Supernatants were ultracentrifuged for 2h at 100.000 x g, after which pellets were resuspended in PBS, filtered (0.22 µm filter) and further ultracentrifuged at 100.000 x g, for 70 min. A last wash with PBS was performed, followed by ultracentrifugation at 100.000 x g, for 70 min. Final pellets were resuspended in sterile PBS for further experiments. Total protein content of EVs was determined using the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). On average, we obtained 45.7 µg/mL serum of human controls, and 46.7 µg/mL serum of AMI patients.

Transmission electron microscopy (TEM)
EVs were fixed with 2% paraformaldehyde (PFA) and deposited on Formvar-carbon coated grids (TAAB Laboratories Equipment, Berks, UK). Samples were washed with PBS and fixed with 1% glutaraldehyde for 5 min. Grids were washed with water, contrasted with an uranyl-oxalate solution pH 7, for 5 min, and transferred to methyl-cellulose -uranyl acetate for 10 min on ice, as previously described [3,4] . Images were collected using a Tecnai G2 Spirit BioTWIN electron microscope (FEI, Oregon, USA) at 80kV.

Nanoparticle tracking analysis (NTA)
EVs were ressuspended in 1 ml of PBS, after which NTA was performed using NanoSight LM 10 instrument (NanoSight Ltd). Analysis settings were optimized and kept constant between samples and each video was analyzed to give the mean size and estimated concentration of particles. Data were processed using NTA 2.2 analytical software.
Densitometric quantification was performed in unsaturated images using Image J (National Institutes of Health).

Real-time RT-PCR
Total RNA was isolated from cells with TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Total RNA was reverse-transcribed using the iScript Select cDNA synthesis kit (Bio-Rad). Real-time RT-PCR reactions were performed using SYBR® Green Supermix (Bio-Rad) and appropriate primers, in a Bio-Rad MyCycler iQ5. Gene expression changes were analyzed using the built-in iQ5 Optical system software. The primers used were the following:    Macrophages stimulated with control EVs protect cardiomyocytes from I/R-induced injury.
Macrophages were incubated with H9c2-derived EV CT or EV ISCH for 24 h, after which the cells were scraped and co-cultured with H9c2 cardiomyoblasts. Cells were subjected to 1h of ischemia,