Abstract 15477: Mitochondrial Drp1 Deficiency in Macrophage Limits Revascularization of Ischemic Muscle via Inflammatory Metabolic Reprograming and Macrophage Polarization

Abstract

Background: In preclinical peripheral artery disease (PAD) models, M2-like anti-inflammatory macrophage polarization, arteriogenesis and angiogenesis are crucial in restoring perfusion recovery and revascularization. Mitochondrial dynamics in macrophage regulates cell metabolism and inflammation. Mitochondrial fission protein, Drp1 GTPase is shown to be involved in pro- or anti-inflammatory macrophage phenotypes in a context-dependent manner. However, role of macrophage Drp1 in reparative neovascularization in experimental PAD model remains unknown. Method and Results: Macrophage-specific Drp1 knockout (MФ-Drp1-KO; LysCre x Drp1 fl/fl) or wild type (WT) mice were used for hindlimb ischemia (HLI), a preclinical PAD model. Drp1 expression was dramatically increased in F4/80 + macrophage in ischemic muscle at day 3 after HLI of WT mice. MФ-Drp1-KO mice showed decreased limb perfusion (78.3%), angiogenesis (53.0 %), arteriogenesis (46.3%), and muscle regeneration (41.9%) after HLI. This was associated with increased pro-inflammatory M1-like MФ (2.2 fold by FACS analysis), p-NFkB and TNFα expression and decreased anti-inflammatory M2-like MФ (2.3 fold by FACS analysis) and p-AMPK in ischemic muscle of MФ-Drp1-KO mice vs. WT mice. In vitro, bone marrow-derived MФ with hypoxia serum starvation (HSS) (in vitro PAD model) induced mitochondrial fission and activated Drp1. Drp1 KO MФ under HSS exhibited increased glycolysis (66.7%, ECAR) via reduced p-AMPK as well as excess mitochondrial (mito)ROS production (2 fold), which in turn increased M1-genes ( Nos2 and Ptgs2 ) and decreased M2-genes ( Arg1 and Retnla ). These responses in Drp1 KO MФ were rescued by AMPK activator AICAR and mitoROS scavenger mitoTEMPO. Finally, conditioned media from HSS-treated Drp1 KO MФ showed increased secretion of pro-inflammatory TNFα and GAPDH and suppressed angiogenic responses of cultured endothelial cells (50%). Conclusion: Drp1 dysfunction in macrophage promotes inflammatory macrophage polarization induced by hyperglycolysis via suppression of anti-inflammatory AMPK as well as excess mitoROS, which limits revascularization in experimental PAD.