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03 - Vito

Extracellular vesicles (EVs) are small vesicles that are released by living cells. EVs carry a specific subset of biomolecules that reflect their originating cell type and condition.

In cardiovascular disease (CVD) the release of EVs from inflamed endothelium into the blood represents a biologically significant communication system. EVs transfer their content to for example monocytes, which is accompanied by the reprogramming of the monocytes. This likely supports tissue repair. Thus, the EV-associated biomolecules contain a rich source of information about the cellular processes in CVD, and are promising for diagnostic and therapeutic purposes.

As EVs from diseased cells circulating in the blood are mostly outnumbered by EVs from healthy cells, workflows are needed for fractionation of disease-specific EV subpopulations. Single EV, multi-parameter-based separation and analysis methods increasingly gain interest for this purpose, since they can provide the sensitivity and specificity of detection needed for clinical diagnostics. A high-resolution flow cytometry instrument for single vesicle sorting and analysis has been installed at VITO (Figure 1) and quality assured protocols have been optimized for fluorescent EV staining, targeting EV subpopulations of interest. This unique infrastructure in Flanders has been used in a pilot study on the effect of the pro-inflammatory stimulus TNF-α on the EV secretion profiles of in vitro cultured human macrophages. A two-fold increase in the number of secreted EVs, as well as a change in protein expression on macrophage-derived EVs has been demonstrated (Figure 2). Sorting of such informative EV subsets will allow to discover and/or verify EV-associated biomolecules specific for CVD conditions, and thus finally aid in the delivery of improved diagnostic tests.

Figure 1: High-resolution flow cytometer BD Influx for small particle sorting and analysis at VITO.

Vito figuur a en b

Figure 2: Effect of tumor necrosis factor-alpha (TNF-α) on the (A) number and (B) CD54/ICAM-1 expression of EVs derived from THP-1 macrophages. Cells were kept overnight in medium containing 2% EV-free serum and supplemented with 30 ng/ml TNF-α. Secreted EVs were isolated using ultracentrifugation, and fluorescently stained with the intra-luminal dye Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and anti-CD54-PECy5 monoclonal antibody. Free dye and antibodies were removed using density centrifugation on a sucrose gradient. Vesicle fractions were diluted (1:20) in PBS and EVs recorded in 30 sec using high-resolution flow cytometry on a BD Influx instrument.

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