Microvascular Extracellular Vesicles: Novel Mediators of Endothelial Communication in Wound Healing and Inflammatory Regulation

Microvascular endothelial cells, which form the innermost lining of the smallest blood vessels, play crucial roles in vascular homeostasis, inflammatory responses, and tissue repair. While extracellular vesicles (EVs) have emerged as important mediators of intercellular communication, those derived from microvascular endothelial cells remain poorly characterized despite their potential significance in regulating local vascular microenvironments. This dissertation addresses critical knowledge gaps through a systematic approach encompassing methodological optimization, comprehensive proteomic characterization, and functional validation. First, an optimized low-serum culture system was developed for the HMEC-1 cell line and established a scalable isolation methodology combining ultracentrifugation with size exclusion chromatography, yielding high-purity EV preparations. Comprehensive proteomic analysis revealed 316 proteins, including 70 not previously documented in microvascular endothelial cell-derived EVs, with significant enrichment for proteins involved in wound healing, angiogenesis regulation, inflammatory response, and cell-cell adhesion. Functional studies demonstrated that these EVs significantly enhance wound closure in both dermal fibroblasts and keratinocytes, with improvements of up to 45% compared to controls. Remarkably, these vesicles exhibited a previously undocumented dual effect on endothelial function—simultaneously upregulating inflammatory markers while enhancing junctional integrity, suggesting a specialized role in maintaining vascular barrier function during inflammatory responses. These findings establish microvascular endothelial cell-derived EVs as complex mediators of intercellular communication capable of coordinating tissue responses during vascular remodeling. Their ability to promote wound healing while preserving barrier function suggests promising therapeutic applications in conditions characterized by impaired tissue repair or endothelial dysfunction. This work provides a foundation for future investigations into the physiological and pathological roles of these vesicles, with potential implications for developing novel approaches to vascular medicine.