HMEC-1 extracellular vesicles as regulators of endothelial cell activation under inflammation

Microvascular endothelial cell-derived extracellular vesicles (EVs) mediate local intercellular communication relevant to wound healing and inflammation, yet their proteomic cargo and functional properties remain poorly characterized. Here, EVs were isolated from human microvascular endothelial cells (HMEC-1) by standard ultracentrifugation (UC Bulk) or ultracentrifugation combined with size exclusion chromatography (UC+SEC) and characterized by nanoparticle tracking analysis, immunoblotting, cryogenic electron microscopy, and label-free mass spectrometry. UC+SEC achieved a 77-fold improvement in particle-to-protein ratio with 70-93% depletion of serum and extracellular matrix contaminants while preserving canonical EV markers (ALIX, CD9). Mass spectrometry identified 673 proteins in UC+SEC versus 336 in UC Bulk, with both preparations enriched in wound healing, hemostasis, and angiogenesis pathways. Despite dramatic purity differences, both isolation methods produced functionally comparable EVs that significantly enhanced dermal fibroblast wound closure. Functional assays on primary human dermal microvascular endothelial cells (HDMECs) revealed that HMEC-1-derived EVs exert inflammation-dependent dual effects on TNF-α pre-treated endothelium: upregulating VCAM-1 expression while simultaneously preserving VE-cadherin-mediated junction integrity. These effects were strictly inflammatory-dependent, with no detectable activity on healthy endothelial cells. This research uncovers a paradoxical phenotype in which microvascular endothelial EVs enhance immune cell recruitment signals while protecting barrier function exclusively under inflammatory conditions, suggesting a regulatory mechanism that may contribute to vascular homeostasis during inflammatory challenges.