Abstract Body (Do not enter title and authors here): Background: Reactive oxygen species (ROS) function as signaling molecules to regulate reparative macrophage polarization essential for ischemia-induced neovascularization and regeneration of skeletal muscle in peripheral arterial disease (PAD). Mitochondrial fission protein Drp1 GTPase is involved in pro- and anti-inflammatory macrophage phenotype and metabolism in a context-dependent manner. However, mechanistic link between macrophage Drp1 and ROS-dependent reparative angiogenesis and muscle regeneration during ischemia is entirely unknown.

Methods and results: Using a mouse Hindlimb ischemia (HLI) model, a preclinical model of PAD, here we show that macrophage-specific Drp1 knockout (KO) mice exhibited significant reduction of blood flow recovery, angiogenesis (CD31+ capillary) and muscle regeneration following HLI. Flow cytometry analysis revealed that these responses were associated with an initial increase in Lys6G+ neutrophils (4-fold), followed by reduction in anti-inflammatory F4/80+CD206+ M2 macrophage (2-fold) in ischemic muscle of Drp1 KO mice at days 3 and 7 post HLI. Mechanistically, Drp1-CysOH formation (sulfenylation), but not Drp1 phosphorylation, was increased in bone marrow (BM) and ischemic muscles after HLI, which was markedly reduced in CRISPR/Cas9-generated “redox-dead” Drp1-C631A (C/A) knock-in mutant (Drp1-C/A) mice. Functionally, BM chimera mice in which BM in wild type (WT) mice is replaced with Drp1-C/A mutant showed impaired limb perfusion recovery (30%), angiogenesis (45%) and muscle regeneration after HLI. In vitro PAD model showed that macrophage exposed to hypoxia serum starvation (HSS) had increased ROS production and Drp1-CysOH formation without Drp1 phosphorylation at 1 h after HSS stimulation. Macrophage isolated from Drp1 KO or Drp1-C/A mice under HSS exhibited inhibition of Drp1-CysOH formation, while increased glycolysis (ECAR, 50%), pro-inflammatory M1-genes and decreased M2-genes (40%).

Conclusion: Cysteine oxidation of Drp1 in macrophage promotes revascularization and regeneration of ischemic muscle by driving reparative metabolic reprogramming and macrophage polarization. This process represents a novel therapeutic target for treatment of PAD.