S-Nitrosylation of Endothelial FK506-Binding Protein 5 Impairs Angiogenesis via PHLPP-AKT Axis in Ischemic Stroke

BACKGROUND: Angiogenesis contributes to vascular repair and functional recovery after ischemic stroke, yet how nitric oxide-mediated S-nitrosylation shapes this response remains unclear. We investigated the role of S-nitrosylation in postischemic angiogenesis and the underlying molecular mechanism. METHODS: S-nitrosylation proteomics was performed in ischemic brain tissue from 8-week-old male mice subjected to transient middle cerebral artery occlusion and in brain microvascular endothelial cells exposed to oxygen-glucose deprivation/reoxygenation. Candidate modification sites were validated by cysteine mutagenesis and biotin-switch assays. Wild-type and C339A-mutant FKBP5 (FK506-binding protein 5) were compared in endothelial angiogenesis assays. Four-week-old male mice received endothelial-targeted adeno-associated virus 9 encoding wild-type or C339A-mutant FKBP5 and underwent transient middle cerebral artery occlusion 4 weeks later. Vascular and neurological outcomes were assessed through day 28. Protein-interaction and signaling analyses defined the downstream mechanism. RESULTS: S-nitrosylated FKBP5, but not total FKBP5, was increased in ischemic brain tissue and oxygen-glucose deprivation/reoxygenation-treated endothelial cells; inducible nitric oxide synthase was an upstream mediator. Mass spectrometry and mutagenesis identified cysteine 339 as the predominant modification site. C339A prevented FKBP5 S-nitrosylation and rescued endothelial proliferation, migration, sprouting, and tube formation after oxygen-glucose deprivation/reoxygenation. In mice, endothelial-targeted expression of FKBP5-C339A promoted peri-infarct angiogenesis and perfusion, reduced tissue injury, and improved chronic sensorimotor recovery. Mechanistically, S-nitrosylation strengthened FKBP5 binding to PHLPP (PH domain leucine-rich repeat protein phosphatase) and reduced AKT (serine/threonine kinase) phosphorylation. C339A weakened this interaction and restored AKT activation, whereas PHLPP inhibition with NSC117079 enhanced AKT signaling and angiogenic responses in vitro. CONCLUSIONS: We identify endothelial FKBP5 as a previously unrecognized regulator of poststroke vascular regeneration and establish S-nitrosylation at cysteine 339 as a molecular switch that restrains angiogenesis and functional recovery through PHLPP-dependent inhibition of AKT signaling. Targeting this modification may offer a strategy to enhance vascular repair after ischemic stroke.