Introduction:

In recent years extracellular vesicles (EVs) have gained increasing attention as intercellular transporters of biologically active molecules. For the generation of EVs, the local accumulation of polar sphingolipids, in particular of ceramides, in the releasing cellular membranes is crucial. This leads to an enrichment of multiple ceramides in EVs. Besides their role in the EV-budding process, ceramides are involved in the induction of apoptosis. Interestingly, the biological effect of different ceramide species depends on the length of their fatty acid chain. In this context, intermediate and long chain ceramides (d18:1-16:0 to d18:1-20:0) have been shown to be particularly potent inductors of apoptosis. In the cardiovascular context, elevated plasma levels of d18:1-16:0 Ceramide (C16) have been associated with an increased risk for cardiovascular events. Under which conditions different sphingolipids are enriched in endothelial cell derived EVs and if the enrichment and a potential transfer to EV recipient cells is biologically relevant, however, has never been assessed.

Methods and Results:

EVs were isolated from human coronary artery endothelial cells (HCAECs) after stimulation with 30 mmol/L glucose or vehicle for 72 h by use of differential centrifugation (1 x 1500 g / 15 min + 2 x 20000 g / 40 min), as previously described by our group. The EVs were characterized by electron microscopy and nanoparticle tracking analysis, which revealed no difference in concentration or size distribution (mean size: 252 ± 24 nm) upon glucose treatment. Total lipid extraction was performed from HCAECs and EVs of the two conditions by use of alkaline hydrolysis and solid phase extraction. The lipidomic analysis of the isolated sphingolipids via Q-TOF MS/MS mass spectrometry showed a significant enrichment of various ceramide species in EVs, among which C16 was the most abundantly present ceramide. Moreover, C16 was significantly enriched in EVs from glucose-damaged HCAECs compared to vehicle treated cells, whereas no difference in the C16 concentration in the EV mother cells was detected after glucose damage. Interestingly, C16 metabolites including the Sphingobase d18:1 and the Sphingomyelin d18:1-16:0 were also enriched in EVs from glucose damaged HCAECS. In order to estimate the toxicity of C16, cell viability was assed via a colorimetric assay (MTT-Assay) upon C16 stimulation. In the performed concentration row, cell viability was significantly reduced at concentrations above 10 µM C16. As a next step, we investigated a potential transfer of C16 via EVs. Therefore, we treated HCAECs with 2 µM and 20 µM C16 for 48 h to allow uptake of the lipid into the cells. Subsequently we isolated EVs from the ceramide treated HCAECs and incubated native HCAECS with the EVs for 24 h. In the MTT assay, we found cell viability to be significantly reduced after treatment with EVs derived from HCAECs after C16 uptake.

Conclusion:

In this study, we showed for the first time that C16 Ceramide and its metabolites are enriched in EVs from Glucose treated HCAECs. C16 accumulation in EVs was independent of EV release and EV size. Additionally, we established that the pro-apoptotic signal of C16 is transferable through EVs. The results point towards a relevant intercellular communication via an EV-based ceramide transfer and highlight the importance of circulating lipids not only as biomarkers but as biologically active molecules.