Hypoxia stimulates blood-brain barrier disruption and systemic appearance of pro-coagulant, brain-derived extracellular vesicles: Implications in transient ischemic attack patients.

Tissue hypoxia and blood-brain barrier (BBB) dysfunction are key features of transient ischaemic attack (TIA) and ischaemic stroke. The neurovascular unit maintains brain homeostasis and coordinates stress responses. Extracellular vesicles (EVs) are emerging as important mediators of cell communication in hypoxia, impacting BBB integrity and enabling bidirectional movement. This study examined EV production by neurovascular cells in normoxia (21% O2) and hypoxia (1% O2) and compared these profiles with circulating EVs in TIA patients. Human brain endothelial cells and astrocytes were cultured under normoxic or hypoxic conditions for up to 24 h. EVs were isolated and analysed via nanoparticle tracking and flow cytometry. A co-culture transwell model assessed BBB permeability under controlled experimental conditions. Circulating EVs from TIA patients, TIA mimics and healthy controls were analysed for cell origin, phenotype and function. Hypoxia increased EV production from endothelial cells, disrupted BBB integrity and promoted EV movement across the barrier. TIA patients had distinct EV profiles, with elevated endothelial-derived (CD9+/CD144+) and astrocyte-derived (CD9+/GFAP+) EVs and proteins. Both cell- and patient-derived EVs enhanced clot formation and resistance to lysis. These findings suggest EVs contribute to post-TIA thrombotic risk. Astrocyte-derived EVs may serve as rapid, cost-effective biomarkers to distinguish TIA from mimics.