Macrophages play a crucial role in promoting perfusion recovery and revascularization after ischemia through antiinflammatory polarization, a process essential for the treatment of peripheral artery disease (PAD). Mitochondrial dynamics, particularly regulated by the fission protein DRP1, are closely linked to macrophage metabolism and inflammation. However, the role of DRP1 in reparative neovascularization remains unexplored. Here, we show that DRP1 expression was increased in F4/80 ⁺ macrophages within ischemic muscle on day 3 after hind limb ischemia (HLI), an animal model of PAD. Mice lacking Drp1 in myeloid cells exhibited impaired limb perfusion recovery, angiogenesis, and muscle regeneration after HLI. These effects were associated with increased proinflammatory M1-like macrophages, p-NF-κB, and TNF-α, and reduced antiinflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1 ^–/– mice. In vitro, Drp1 -deficient macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction, and excessive mitochondrial ROS production, resulting in increased proinflammatory M1-gene and reduced antiinflammatory M2-gene expression. Conditioned media from HSS-treated Drp1 ^–/– macrophages exhibited increased proinflammatory cytokine secretion, leading to suppressed angiogenesis in endothelial cells. Thus, macrophage DRP1 deficiency under ischemia drives proinflammatory metabolic reprogramming and macrophage polarization, limiting revascularization in experimental PAD.

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.