Angiogenesis is essential for development and tissue repair after ischemia. Reactive oxygen species (ROS) act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly rely on aerobic glycolysis for energy production. However, how redox signaling couples to endothelial metabolism remains unclear. Here, we identify endothelial Drp1 as a redox sensor that links VEGF-induced H₂O₂ signaling to metabolic reprogramming and angiogenesis. Loss of Drp1 in ECs suppresses VEGF-driven angiogenic responses. Mechanistically, VEGF rapidly induces NOX4-dependent sulfenylation of Drp1 at Cys⁶⁴⁴, promoting disulfide bond with the metabolic kinase AMPK and subsequent oxidation of AMPK at Cys^299/304 via mitochondrial fission-derived ROS. This pathway enhances endothelial glycolysis and angiogenesis. In vivo, mice with endothelial Drp1 deficiency or CRISPR-engineered redox-dead Drp1 (Cys to Ala) knock-in exhibit impaired retinal angiogenesis and post-ischemic neovascularization. Thus, endothelial Drp1 integrates mitochondrial redox signaling with glycolysis through cysteine oxidation-mediated Drp1-AMPK redox relay, thereby driving reparative neovascularization.

Reparative macrophage polarization and macrophage-derived reactive oxygen species (ROS) are required for ischemia-induced revascularization in peripheral artery disease (PAD). Our previous study showed that mitochondrial fission protein dynamin-related protein 1 (DRP1) promotes reparative polarization and metabolic reprogramming in macrophages and post-ischemic neovascularization. However, the redox-dependent mechanism governing DRP1 activation in this context remains elusive. Here, using a mouse hindlimb ischemia (HLI) model of PAD, we identify cysteine sulfenylation (CysOH) of DRP1 as a critical redox modification induced in ischemic bone marrow (BM)-derived cells. BM chimeric mice reconstituted with CRISPR/Cas9-generated “redox-dead” DRP1-C631A knock-in mutant (Drp1^C/A) BM exhibited markedly reduced limb perfusion recovery and CD31⁺ capillary density in ischemic muscles following HLI. These defects were associated with enhanced Ly6G⁺ neutrophil accumulation, pro-inflammatory F4/80⁺CD80⁺ M1-like macrophages and reduced anti-inflammatory F4/80⁺CD206⁺ M2-like macrophages in ischemic muscle. Mechanistically, using an in vitro PAD model, hypoxia serum starvation (HSS) rapidly induced NADPH oxidase 2-dependent cytosolic ROS production and DRP1-CysOH formation in wild-type macrophages. In contrast, Drp1^C/A macrophages failed to undergo DRP1-CysOH-dependent mitochondrial fission under HSS, resulting in aberrant metabolic reprogramming characterized by enhanced glycolysis and mitochondrial ROS, pro-inflammatory p-NF-κB and M1-genes, and suppressed anti-inflammatory p-AMPK, efferocytosis and M2-genes. Thus, our findings establish DRP1 sulfenylation as a previously unrecognized redox-sensing mechanism that links ischemia-induced ROS to reparative macrophage reprogramming and revascularization, identifying a novel therapeutic target for PAD.