Abstract

Extracellular vesicles (EVs) such as exosomes are nano-sized vesicles that carry proteins and miRNAs and can transmit signals between cells. We hypothesized that exosomes from endothelial cells can transmit protective signals to cardiomyocytes. Co-culture of primary adult rat cardiomyocytes with normoxic HUVEC cells separated by a cell-impermeable membrane reduced the percentage of cardiomyocyte death following simulated ischaemia and reperfusion (sIR) from 80 ± 11% to 51 ± 4% (P < 0.05; N = 5). When EVs were removed from the HUVEC-conditioned medium it was no longer protective. Exosomes were purified from HUVEC-conditioned medium using differential centrifugation and characterized by nanoparticle tracking analysis, electron microscopy, and flow cytometry. Pre-incubation of cardiomyocytes with HUVEC exosomes reduced the percentage of cell death after sIR from 88 ± 4% to 55 ± 3% (P < 0.05; N = 3). This protection required ERK1/2 activity as it was prevented by inhibitors PD98059 and U0126. Ischaemic preconditioning caused about ~3-fold higher rate of exosome production from HUVEC and from isolated, perfused rat hearts. This increase resulted in significantly greater protection against sIR in cardiomyocytes. In conclusion, exosomes released from endothelial cells can confer resistance to sIR injury in cardiomyocytes via the activation of the ERK1/2 MAPK signalling pathway, and may contribute to IPC.

Highlights

  • Ischaemia and reperfusion injury (IRI) is a major contributing factor to the death of cardiomyocytes that occurs during myocardial infarction[1,2]

  • We used a co-culture model with both human umbilical vein endothelial cells (HUVEC) and primary adult rat cardiomyocytes separated by a cell-impermeable membrane, to investigate whether endothelial cells release exosomes that can stimulate cardioprotection in recipient cardiomyocytes, whether Ischaemic preconditioning (IPC) increases the release of these nano-sized vesicles, and whether these might contribute to preconditioning

  • After 45 min exposure, the insert containing the endothelial cells was removed, and the cardiomyocytes were incubated in a buffer simulating ischaemia and placed in a hypoxic chamber for 2.5 h followed by re-oxygenation for 30 min (H/R) (Fig. 1A)

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Summary

Introduction

Ischaemia and reperfusion injury (IRI) is a major contributing factor to the death of cardiomyocytes that occurs during myocardial infarction[1,2]. Exosomes are nano-sized extracellular vesicle (EVs) released by most cell types[10,11,12,13]. Exosome administration results in long-term improvement in ventricular function via various pathways including the stimulation of angiogenesis, immunosuppression, and potentially the activation of regenerative pathways[12,13]. Large numbers of exosomes are continually released into the circulation by different cell types including platelets, erythrocytes, leukocytes and endothelial cells, and these may contribute to cardiovascular protection. We used a co-culture model with both human umbilical vein endothelial cells (HUVEC) and primary adult rat cardiomyocytes separated by a cell-impermeable membrane, to investigate whether endothelial cells release exosomes that can stimulate cardioprotection in recipient cardiomyocytes, whether IPC increases the release of these nano-sized vesicles, and whether these might contribute to preconditioning

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