Abstract Body (Do not enter title and authors here): Background: Macrophage plays a critical role in promoting perfusion recovery and revascularization through anti-inflammatory polarization and metabolic reprogramming, required for treating peripheral artery disease (PAD). Copper (Cu) is an essential micronutrient, and dysregulated Cu levels contribute to macrophage dysfunction. However, the specific role of macrophage-specific Cu uptake transporter CTR1 in post-ischemic neovascularization remains unexplored.
Methods and Results: Using a mouse Hindlimb ischemia (HLI) model, a preclinical model of PAD, we here demonstrate that CTR1 expression was dramatically increased in F4/80⁺ macrophages in ischemic muscle at day 3 after HLI of WT mice. Myeloid-specific Ctr1 deficient mice exhibit impaired limb perfusion recovery (45%), reduced angiogenesis CD31⁺ capillaries (90%) and a-SMA⁺ arterioles (86%) following HLI. Flow cytometric analysis of ischemic muscles from Ctr1^KO macrophage mice vs wild-type (WT) mice after HLI revealed 39% decrease in numbers of anti-inflammatory M2 (F4/80⁺/CD64⁺/CD206⁺)-macrophage and 50% increase in proinflammatory M1 (F480⁺/CD64⁺/CD80⁺)-macrophage, without affecting numbers of CD11b⁺/Ly6C^hi monocytes and mature F480⁺/CD64⁺ macrophages. Mechanistically, under hypoxia serum starvation (HSS), an in vitro PAD model, expression of CTR1 and Cu intake was significantly increased in WT bone marrow derived macrophages (BMDM) by 2h of HSS stimulation. Ctr1-deficient BMDM (Ctr1^KO macrophage) exhibited enhanced glycolysis (43%) along with 2-fold increased expression of proinflammatory and cytosolic DNA sensor cGAS-STING pathway proteins. This shift led to increased proinflammatory M1-gene and reduced anti-inflammatory M2-gene expression. This responses in Ctr1^KO macrophage were rescued by STING inhibitor. Finally, conditioned media from HSS-treated Ctr1^KO macrophage vs WT macrophage suppressed angiogenic responses of endothelial cells by 66% upon wound scratch stimulation.
Conclusion: In summary, macrophage CTR1 deficiency under ischemic conditions promotes cGAS-STING pathway, pro-inflammatory metabolic reprogramming and macrophage polarization, leading to impaired revascularization in experimental PAD.