The hypoxic signature response of cells and their derived Extracellular Vesicles in the Neurovascular Unit and their role in Ischaemic Disease

Stroke remains a major cause of mortality and long-term disability in the UK, with approximately 100,000 cases annually, over 85% of which are ischaemic. Tissue hypoxia arising from reduced cerebral blood flow disrupts the blood–brain barrier (BBB) and promotes bidirectional leakage between the vasculature and brain parenchyma. Extracellular vesicles (EVs) are small membrane-bound particles capable of crossing the BBB, and endothelial derived EVs are known to increase following ischaemic injury. This thesis investigated the “hypoxic signature response” of neurovascular unit (NVU) cells and their EVs and explored their contribution to BBB dysfunction and ischaemic disease.The primary objective was to characterise EV production in human brain endothelial cells (HBECs) and astrocytes under short- and long-term hypoxia. A secondary aim was to establish a robust in vitro BBB model using these cell types and assess barrier disruption and EV translocation under hypoxic conditions, simulating transient ischaemic attack (TIA) and ischaemic stroke (IS). The pathological potential of EVs generated under hypoxia was examined using functional coagulation assays. A further objective was to compare in-vitro findings with circulating EV profiles in healthy individuals and patients who had recently experienced a TIA, utilising data from the PREDICT-EV clinical study. Hypoxia significantly disrupted BBB integrity in vitro and markedly increased EV production in HBECs, whereas astrocyte EV production remained unchanged. Hypoxic conditions also enhanced the translocation of EVs across the in vitro BBB, demonstrating increased leakage during barrier compromise. Functional assays revealed that astrocyte-derived EVs accelerated clot formation, indicating a potential prothrombotic influence within the hypoxic NVU environment. In the clinical cohort, GFAP⁺ astrocyte-derived EVs were elevated in patients following TIA compared to healthy controls and critically were higher in confirmed TIA cases than in TIA mimics, suggesting diagnostic potential for distinguishing true TIA events in the clinical setting.Collectively, this thesis establishes a mechanistic link between hypoxia-induced BBB disruption, altered EV dynamics, and coagulation pathway activation. By integrating in vitro modelling with clinical EV profiling, the work identifies GFAP⁺ EVs as a promising biomarker candidate for improving TIA diagnosis and offers new insight into NVU-specific hypoxic responses relevant to early stroke pathophysiology.