Stroke severity shapes extracellular vesicle profiles and their impact on the cerebral endothelial cells

Ischaemic stroke is a leading cause of death and disability. Circulating extracellular vesicles (EVs) post-stroke may help brain endothelial cells (BECs) counter ischaemic injury. However data on how EVs from ischaemic stroke patients, considering injury severity, affect these cells are limited. The aims were to characterize the inflammatory and angiogenic components of circulating EVs in acute ischaemic stroke patients, considering stroke severity, and to investigate whether these circulating EVs differentially influence the proangiogenic properties and blood-brain barrier (BBB) integrity of human BECs. Eighteen ischaemic stroke patients (acute phase: 24-48 h) and nine controls matched by age, sex, and blood pressure were studied. Stroke severity was classified as severe (n = 9) or mild (n = 9). Plasma EVs were analysed for size, concentration, and protein markers (CD63, Alix, CD81, TSG101, HSP70), as well as proinflammatory and angiogenic proteins. EV uptake, cell viability, proangiogenic capacity, electrical resistance [TEER (transendothelial electrical resistance)], and dextran-70 kD permeability were assessed using human brain microvascular endothelial cells (hCMEC/D3). Stroke patients had lower EV concentrations than controls (p = 0.075), with mild-stroke patients having the smallest EVs. Stroke-derived EVs had higher levels of interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), nitrotyrosine, and vascular endothelial growth factor (VEGF) but lower placental growth factor (PLGF) compared to controls. IL-6 was higher in mild strokes (p = 0.0025), and VEGF was higher in severe strokes (p = 0.048). EVs from severe-stroke cases enhanced proangiogenic capacity and minimally disrupted the BBB. Stroke severity influences EV number, size, and composition. EVs from severe strokes may promote BBB restoration and cerebral angiogenesis, suggesting their role in intercellular communication and homeostasis in ischaemic tissue. KEY POINTS: Ischaemic stroke is one of the leading causes of death worldwide. After an ischaemic stroke several physiological processes are triggered to recover the injured tissue. Increasing evidence has suggested that extracellular vesicles (EVs) present in the bloodstream could play a role in brain recovery, but their specific impact, especially concerning stroke severity, was unclear. This study demonstrates that plasma-derived EVs from first-ever ischaemic stroke patients have distinctive characteristics and effects over brain angiogenesis and blood-brain barrier (BBB) integrity. Our study proposes that circulating EVs from patients with severe stroke may carry protective factors to initiate brain endothelial cell recovery after acute episodes. These findings underscore the role of EVs as potential effectors of BBB recovery and biomarkers in severe ischaemic stroke.