Photobiomodulation restores blood–brain barrier integrity after hypoxia via endothelial von Willebrand factor modulation in a humanised tricellular transwell model

) and applied three PBM treatments during recovery. Hypoxia significantly reduced transendothelial electrical resistance (TEER), whereas PBM restored barrier function in endothelial monocultures and tri-cultures. Endothelial cells exhibited the most pronounced hypoxic response, characterised by increased expression of hypoxia-inducible factor-1α (HIF-1α), plasminogen activator inhibitor-1 and von Willebrand factor (vWF), all attenuated by PBM. Importantly, small interfering RNA-mediated knockdown of vWF partially recapitulated PBM-induced restoration of barrier integrity, identifying endothelial vWF as a mediator of recovery. PBM also reduced reactive oxygen species in hypoxic astrocytes and pericytes, indicating co-ordinated multicellular modulation. Together, these findings demonstrate that PBM restores BBB integrity following hypoxic insult by modulating endothelial thrombo-inflammatory signalling at the same time as reducing oxidative stress in glial cells. Rather than acting as a non-specific cytoprotective stimulus, PBM engages molecular pathways linked to endothelial activation. This work establishes a mechanistically informed platform for investigating BBB repair and highlights PBM as a strategy to protect vascular integrity in hypoxia-associated neurological disorders. KEY POINTS: Hypoxia is a major driver of blood-brain barrier (BBB) dysfunction, yet there are currently no targeted therapies that directly restore barrier integrity. Photobiomodulation (PBM) is a non-invasive low-level light intervention known to facilitate mitochondrial function and cellular stress responses. In a human in vitro BBB model, repeated PBM treatment restored transendothelial electrical resistance (TEER) 24 and 48 h after hypoxic injury, with endothelial rescue linked to downregulation of von Willebrand factor (vWF). PBM modulated oxidative stress, hypoxia signalling and thrombo-inflammatory pathways across endothelial cells, astrocytes and pericytes. These findings support PBM-driven modulation of endothelial signalling as a potential strategy to restore BBB integrity in hypoxia-associated neurological conditions.