P2583Endothelial cell-derived extracellular vesicles promote M2 polarization of THP-1 cells

Abstract

Abstract Introduction The intercellular transfer of biologically active molecules in extracellular vesicles (EVs) has recently been discovered as an important mechanism, which regulates cardiovascular health and disease. In the context of atherosclerosis, endothelial cell-derived EVs have been shown to modulate the phenotype of EV recipient cells in a relevant manner. Under pro-atherogenic conditions e.g. hyperglycemia, the export of numerous signal molecules in EVs is altered and so are EV-dependent effects on recipient cells. While the effect of endothelial-cell derived EVs on other endothelial cells and vascular smooth muscle cells is well characterized, little is known about the vesicle-based interaction of endothelial cells and monocytes under pro-atherogenic conditions. This is particularly relevant as monocytes are crucial modulators of vascular regeneration and inflammation. Our aim was therefore to investigate, whether EVs from endothelial cells have a significant effect on the differentiation and polarization of monocytes and how this process is affected by pathologic conditions as mentioned above. Methods and results Human Coronary Arterial Endothelial Cells (HCAECs) were cultured in high-Glucose-medium (30 mM) for 72h. PBS was used as a control. Large EVs were isolated from the culture supernatant by differential centrifugation (1 x 1500 g / 15 min + 2 x 2ehz748.0909 g / 40 min). The harvested large EVs were characterized by Immunoblotting, Nanoparticle Tracking Analysis as well as Transmission electron microscopy and were shown to be mostly between 80 and 500 nm in size. Specific surface markers including Annexin V and Flotillin-1 were highly enriched in the isolated EVs. The EVs were used for co-culture with THP-1 cells with and without previous phorbol-12-myristate-13-acetate (PMA) stimulation. After 4h as well as after 24 h of incubation with EVS, total RNA was isolated from the THP-1 cells and qPCR was performed to assess polarization towards M1 by TNF-α gene expression or M2 by IL-10 expression. While EV treatment of THP-1 cells without previous PMA-stimulation showed no measurable effect, a significant decrease in the expression of TNF-α was detected after 4 h of treatment from Glucose injured HCAECs. Similar results were obtained without glucose stimulation, the most significant reduction of TNF-α expression however was seen at 24 h. In regard to IL-10 no significant expression changes were detected in EV treated THP-1 cells. Conclusion We showed that glucose injury does not relevantly affect vesicle release or size. Furthermore, endothelial cell derived EVs cause a reduction of TNF-α expression, which indicates a polarization towards an M2 macrophage phenotype, irrespective of prior hyperglycaemic injury. As the M2 phenotype has been described as pro-regenerative, we conclude that endothelial cell derived EVs can exert a protective function during the invasion of monocytes in cardiovascular disease and remodeling.