Surface N-Glycosylation Dictates MSC-EV Uptake and CCR2-Driven Monocyte Recruitment to Inflamed Endothelium Under Shear Flow.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) hold promising potential for immunomodulation and tissue regeneration, especially for treating ischemia-reperfusion injury (IRI). However, their therapeutic use is hindered by challenges in tissue targeting and incomplete understanding of their functional mechanisms. Upon systemic administration, MSC-EV first encounter IRI-activated endothelium, whose response would determine the nature and extent of subsequent immune cell infiltration and inflammation, with monocytes shaping the type of immune response. Our previous work showed that surface N-glycans are key for MSC-EV interaction with endothelial cells. Here, we investigate the role of N-glycosylation in MSC-EV interaction with TNF-activated endothelial cells and subsequent monocyte rolling and adhesion. Using static and dynamic flow in vitro models, we show that depletion of surface N-glycans specifically targeted MSC-EV to inflamed endothelium. Conversely, while intact MSC-EV inhibited monocyte adhesion and fostered MSC recruitment to endothelial cells, this was lost upon N-glycan depletion. Finally, our results suggest that MSC-EV-mediated modulation of the MCP-1/CCR2 interaction may underlie the reduced monocyte infiltration into inflamed endothelium. These findings highlight the role of N-glycosylation in the immunomodulatory activity of MSC-EV, offering a potential mechanism of action and suggesting that glycoengineered MSC-EV may enhance their therapeutic effectiveness in inflammatory and ischemic conditions.