DAPI staining was used to visualize nuclei. The cultures were then washed 3 times with PBS and fixed with cold (− 20 ☌) acetone for 5 min at − 20 ☌. A medium containing EVs was added for 24 h of incubation. The pellet containing PKH26-labeled EVs was resuspended in 1 ml of cell culture medium.įor confocal examinations, TIME cells and HUVECs were cultured in glass bottom dishes to reach 50% confluence. After this step, the supernatant was discarded and the pellet of EVs was washed in 1 ml of PBS and ultracentrifuged once more.
Then, EVs were blocked with 50 μl of 1% BSA, dissolved in 900 μl of phosphate buffered saline (PBS) and ultracentrifuged under the same conditions. A sample without EVs was used as a negative control to determine any carryover of PKH26 dye. In brief, 0.1 μl of PKH26 was added to a pellet of EVs in a total of 50 μl of diluent C and incubated for 20 min at room temperature. The proposed approach should be suitable to investigate EV fate in further experiments.Ĭellular uptake of endothelial-derived EVsĮndothelial-derived EVs were labelled with PKH26 as previously described with a minor modification. We also wanted to show the intracellular distribution of endothelial-derived EVs in the targeted endothelial cells to gain a better insight into EV trafficking mechanisms. The aim of our study was to evaluate whether endothelial-derived EVs might be taken up by endothelial cells and to assess whether they can act as paracrine factors for neighboring cells in further studies. After internalization, endothelial-derived exosomes have beneficial or detrimental effects on the targeted endothelial cells by improving their angiogenic properties or maintaining a pathogenic phenotype. They also respond to different pro- and anti-proinflammatory signals. By secreting EVs, they contribute to both coagulation and fibrinolysis.
Įndothelial cells are vascular cells with paracrine and autocrine properties. Clathrin-independent endocytosis with some contribution of lipid transfer seems to be most likely. For example, it is not known how they are taken up by endothelial cells. However, the precise molecular uptake mechanisms and cellular fate of EVs are still unknown. The glycoproteins (e.g., HSPG ) and proteins (e.g., tetraspanins, integrins ) on the surfaces of EVs and their target cells are known to determine the uptake mechanism. EVs might be internalized by cells in a variety of endocytic pathways (e.g., clathrin-dependent endocytosis ) and clathrin-independent pathways (e.g., macropinocytosis, phagocytosis, caveolin-mediated uptake, lipid raft-mediated internalization ). Several experimental studies have shown that EVs contain various proteins, bioactive lipids, miRNAs and even mRNAs, and that they transfer them between cells contributing to cell-to-cell communication. The formation of apoptotic bodies takes place at the end of the apoptosis process. Exosomes originate from multi-vesicular bodies (MVBs), whereas ectosomes are released from the cell membrane in a shedding process. ĮVs also vary in the way they are produced and released. Based on their sizes and biological properties, they are divided into three groups: exosomes, which range between 50 and 100 nm ectosomes, which range between 1 nm in diameter and apoptotic bodies, which are over 1000 nm in diameter.
Extracellular vesicles (EVs) are nanosized, membrane-derived vesicles.