1) The EVICARE team-members have established the functional consequences of EV therapeutics in the setting of acute myocardial damage. Several important detrimental cardiac consequences upon injury are being affected, including local cell division, immune cell activation and connective tissue formation. Bio-distribution studies in which evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles demonstrated an effective cardiac retention.
2) To explore and determine functional targets of our EVs, (phosphor)proteomics was performed and they established a series of knock-down vesicles to study the mechanisms of target cell stimulation in vitro. PAPP-A was shown to be responsible for promoting angiogenesis through both associated- and co-isolated proteins. For down-stream functional testing, multiple endothelial cell, immune-cell responses and fibrosis assays were established to study their behaviour. It was recently demonstrated that functional intramyocardial injection of cells into the heart is mediated by macrophage polarization. To study and explore modulators of EV release from the diseased heart, e.g. cardiomyocytes, we created a monitoring system that allows real-time and quantitative evaluation of cardiomyocyte-specific EV release.
3) To determine if EV release by progenitor cells can be stimulated, we explored the maximum effect that can be expected by interfering with endolysosomal trafficking. Although this was successful, we also realized that EV release can only be stimulated in a limited way and therefore focussed on other aspects to improve their work load. Production protocols for EV upscaling and isolation were established, but subsequent evaluation of functional in vivo effects demonstrated a reduced effectiveness. This was caused by either the need for both EV-associated as co-isolated proteins that were less present in the optimized protocols or a drift in the used cell-lines. Pro-angiogenic effects were still observed and detailed mechanistic studies executed. In associated work and to overcome the reduced effectiveness, we developed a technology of protein delivery through EVs (TOP-EVs) as a versatile platform for intracellular protein delivery. Functional siRNA Delivery was further explored by Extracellular Vesicle-Liposome Hybrid Nanoparticles. Hereby, we developed novel directions for delivery strategies in which EV-based technologies will be used to move forward the promising therapeutic effects.
