Inhibition of endothelial ALOX12 mitigates cerebral ischemia-reperfusion injury by suppressing 12-HETE.

In acute ischemic stroke (AIS), the efficacy of reperfusion therapy is limited by cerebral ischemia-reperfusion (I/R) injury, in which endothelial dysfunction and angiocrine lipid mediators drive blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative stress. Among these mediators, ALOX12-derived 12-HETE has emerged as a key effector, yet its cellular origin and downstream impact in cerebral I/R remain incompletely defined. In this study, we combined single-cell RNA sequencing and untargeted metabolomics with mechanistic validation in a mouse transient middle cerebral artery occlusion (tMCAO) model and OGD/R-based cell systems to define ALOX12-12-HETE dysregulation after cerebral I/R. Endothelial localization and functional contribution were further assessed by microvessel-parenchyma fractionation, the selective ALOX12 inhibitor ML355, and endothelial-targeted AAV-BR1-mediated Alox12 knockdown. In parallel, plasma 12-HETE levels were measured in propensity score-matched AIS patients and healthy controls. Single-cell analysis identified endothelial cells as a major site of ALOX12 induction after I/R, alongside increased 12-HETE accumulation. Functionally, 12-HETE acted as a deleterious angiocrine mediator that impaired endothelial tight-junction integrity, promoted pro-inflammatory microglial activation, and exacerbated neuronal oxidative injury, as reflected by mitochondrial ROS accumulation, antioxidant depletion, and apoptosis. Pharmacological inhibition of ALOX12 with ML355 attenuated neurovascular injury, improved survival, and restored redox homeostasis, while endothelial-targeted Alox12 knockdown produced comparable protective effects. At the signaling level, ML355 was associated with reduced KEAP1 expression, enhanced nuclear NRF2 accumulation, and increased HO-1 expression, consistent with partial restoration of antioxidant signaling. Clinically, plasma 12-HETE levels were significantly elevated in AIS patients and positively correlated with stroke severity (NIHSS). Together, these findings support endothelial ALOX12-12-HETE signaling as a redox-active angiocrine pathway contributing to multicellular neurovascular injury after cerebral I/R and highlight ALOX12 inhibition as a potential therapeutic strategy.