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

A functional blood-brain barrier (BBB) is essential for CNS homeostasis, and its disruption is an early feature of both acute brain injury and chronic neurodegenerative disorders. Hypoxia induces BBB breakdown by triggering endothelial dysfunction, oxidative stress, metabolic dysregulation and thrombo-inflammatory signalling that compromise barrier integrity. However, strategies that restore BBB function remain limited. Here, we investigated whether photobiomodulation (PBM), a non-invasive light therapy, can rescue BBB dysfunction following acute hypoxic stress. Using a multicellular in vitro BBB model comprising immortalised human brain microvascular endothelial cells, pericytes and astrocytes, we induced hypoxic injury (6 h, 1% O2) 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.