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

Cetaceans are at elevated risk of accumulating persistent and lipophilic environmental contaminants due to their longevity and high proportion of body fat. Despite this, there is a paucity of taxa-specific chemical effect data, in part due to the ethical and logistical constraints in working with highly mobile aquatic species. Advances in cetacean cell culture have opened the door to the application of mainstream in vitro toxicological effect assessment approaches. Image-based cell profiling is a high-throughput, microscopy-based system commonly applied in drug development. It permits the analysis of the xenobiotic effect on multiple cell organelles simultaneously, hereby flagging its potential utility in the evaluation of chemical toxicodynamics. Here we exposed immortalized humpback whale skin fibroblasts (HuWaTERT) to six priority environmental contaminants known to accumulate in the Southern Ocean food web, in order to explore their subcellular organelle responses. Results revealed chemical-dependent modulation of mitochondrial texture, with the lowest observed effect concentrations for chlorpyrifos, dieldrin, trifluralin, and p,p’-dichlorodiphenyldichloroethane of 0.3, 4.1, 9.3, and 19.8 nM, respectively. By contrast, no significant changes were observed upon exposure to endosulfan and lindane. This study contributes the first fixed mitochondrial images of HuWaTERT and constitutes novel, taxa-specific chemical effect data in support of evidence-based conservation policy and management.