In Vitro Analysis of TWEAK/Fn14 Axis in the Blood–Brain Barrier Models during Oxygen–Glucose Deprivation and Reoxygenation

Upregulation of tumor necrosis factor-like weak apoptosis-inducing factor (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) was observed in stroke patients and murine models, contributing to neuronal apoptosis and blood-brain barrier (BBB) disruption. This study aimed to investigate the TWEAK/Fn14 signaling axis in cerebral ischemia and reperfusion using different in vitro oxygen-glucose deprivation (OGD) durations and cellular models. Western blot and RT-qPCR were used to evaluate TWEAK/Fn14 expression in monocultures, co-cultures, and triple-cultures of human immortalized endothelial cells, pericytes, and astrocytes. Six OGD conditions were tested: 4, 8, and 16 h, with or without 24 h reoxygenation. BBB model integrity was evaluated by analyzing occludin, zonula occludens-1, and VE-cadherin. A significant, duration-dependent downregulation of Fn14 was observed in monocultures after OGD (up to 85%, p < 0.05-p < 0.001), with partial recovery after 24 h reoxygenation (p < 0.05). TWEAK levels remained stable with minor fluctuations. Similar Fn14 reductions were seen in co- and triple-cultures (p < 0.01), followed by recovery. Endothelial biomarkers exhibited an initial stress response post-OGD, followed by recovery during reoxygenation. In conclusion, TWEAK remains stable during ischemia without immune cells, while Fn14 is downregulated during OGD and recovers after reoxygenation, indicating time-dependent roles in ischemic response and repair. The findings indicate a time-dependent regulation of Fn14 under ischemic conditions in vitro, highlighting its role in BBB stress and recovery. Nevertheless, further preclinical studies are needed to establish its therapeutic potential.