Myeloid Drp1 Deficiency Limits Revascularization in Ischemic Muscles via Inflammatory Macrophage Polarization and Metabolic Reprograming

Abstract

In the preclinical model of peripheral arterial disease (PAD), M2-like anti-inflammatory macrophage polarization and angiogenesis are required for revascularization. The regulation of cell metabolism and inflammation in macrophages is tightly linked to mitochondrial dynamics. Drp1, a mitochondrial fission protein, has shown context-dependent macrophage phenotypes with both pro- and anti-inflammatory characteristics. However, the role of macrophage Drp1 in reparative neovascularization remains unexplored. Here we show that Drp1 expression was significantly increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Myeloid-specific Drp1-/- mice exhibited reduced limb perfusion recovery, angiogenesis and muscle regeneration after HLI. These effects were associated with an increase in pro-inflammatory M1-like macrophages, p-NFkB and TNF, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1-/- mice. In vitro, Drp1-/- 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, resulting in increased M1-gene and reduced M2-gene expression. Conditioned media from HSS-treated Drp1-/- macrophages exhibited increased secretion of pro-inflammatory cytokines and suppressed angiogenic responses in cultured endothelial cells. Thus, Drp1 deficiency in macrophages under ischemia drives inflammatory metabolic reprogramming and macrophage polarization, thereby limiting revascularization in experimental PAD.