Background Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. Methods and Results A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1–16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. Conclusion lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides. Graphical abstract
Introduction Extracellular vesicles (EVs) have recently been identified as important intercellular transporters of biologically active molecules. Besides microRNAs, which have been shown to mediate an important part of the EV function, various sphingolipids have been shown to be exported into EVs. Among these sphingolipids, ceramides have been gained interest in the cardiovascular context, because elevated plasma levels of d18:1–16:0 Ceramide (C16) have been shown to correlate with an increased risk for cardiovascular events. How ceramides are enriched in endothelial cell derived-EVs and if they are transferred to EV recipient cells to exert specific biological functions is currently unknown. Methods and results Endothelial cell derived large EVs were isolated from human coronary artery endothelial cells (HCAECs) via differential centrifugation of the culture supernatant (1 x 1500 g / 15 min + 2 x 20,000 g / 40 min). EVs were characterized by immunoblotting, electron microscopy and nanoparticle tracking analysis (Size: 252±24 nm). Sphingolipids were extracted from HCAECs and EVs (with and without glycemic injury by 30mmol/L glucose for 72 h) by solid phase extraction and analysed via Q-TOF MS/MS mass spectrometry. Lipidomic analysis revealed an enrichment of all sphingolipid classes in EVs, including C16, which was most abundantly present in EVs. As a next step, we tested if ceramides can be transferred between HCAECs by EVs. Therefore, HCAECs were incubated with NBD-labeled ceramide. NBD-Ceramide uptake into HCAECs and vesicular transfer to native HCAECs were confirmed by fluorescence microscopic imaging. Importantly, the transfer of NBD-Ceramide was abrogated if the vesicles were degraded in Triton-X 1% before incubation with EV recipient cells, which confirms that the transfer is vesicle dependent. Additionally, uptake of C16 into HCAECs and release into EVs was confirmed via mass spectrometry. Viability of C16 treated HCAECs was significantly reduced at concentrations above 5 μM of C16 in an MTT-based assay. In order to assess if the increased vesicular packing and transfer of Ceramides also reduces viability of EV recipient cells, native HCAECs were incubated with EVs from HCAECs which were treated with 2 μM and 20 μM C16. Treatment with C16 enriched vesicles lead to a significant reduction of EV recipient cell viability in a dose dependant manner. Conclusion In the present study, we found that C16 is enriched in EVs and that C16 is transferred through EVs among HCAECs. Depending on the amount of transferred C16, this process reduces the viability of native EV recipient HCAECs. Furthermore, vesicular C16 export is increased after glycemic injury. In comparison to the amounts of free C16, which are necessary to induce a similar reduction in HCAEC viability, the amount of EV transferred C16 is negliable. Hence, vesicular packaging results in a high bioavailability of ceramides leading to a relevant regulation of endothelial cell death. Acknowledgement/Funding BONFOR Progamm University Bonn
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