Myeloid DRP1 deficiency limits revascularization in ischemic muscles via inflammatory macrophage polarization and metabolic reprogramming
Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai
Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai
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Research Article Angiogenesis Inflammation

Myeloid DRP1 deficiency limits revascularization in ischemic muscles via inflammatory macrophage polarization and metabolic reprogramming

Abstract

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.

Authors

Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai

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Figure 1

Myeloid Drp1KO mice exhibited impaired reparative neovascularization via reducing angiogenesis and arteriogenesis in response to ischemia.

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Myeloid Drp1KO mice exhibited impaired reparative neovascularization via...
(A) Immunofluorescence analysis for DRP1 (red) or F4/80+ macrophage (green) expression and their colocalization in non-ischemic and ischemic gastrocnemius (GC) muscles on day 3 after HLI. Scale bars: 10 μm. Inset shows region of interest at the same magnification. (B) Schematic representation of breeding strategy for generating myeloid-specific Drp1–/– (MɸDrp1KO) mice by crossing LysM-Cre mice with Drp1fl/fl mice. (C) Immunoblotting (IB) for DRP1 protein expression in bone marrow-derived macrophages (BMDMs), peritoneal macrophages (PMs), lungs, and hearts isolated from Drp1fl/fl (WT) and MɸDrp1KO (MɸKO) mice. (D) Upper panels show representative laser Doppler images of legs on day 0 and day 21. Lower panels show the blood flow recovery after HLI as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in Drp1fl/fl, LysM-Cre, and MɸKO mice (n = 8–12 mice per group, 2-way ANOVA followed by Tukey’s multiple-comparison test). *P < 0.05 for MɸDKO vs. Drp1fl/fl (WT); #P < 0.05, ##P < 0.01 for MɸDKO vs. LysM-Cre+/– Drp1+/+ (WT). (E) Immunohistochemical analysis for CD31 staining (capillary density), n = 4 mice per group, unpaired, 2-tailed Student’s t test. (F) Immunofluorescence analysis of αSMA+ staining (arterioles) in ischemic and non-ischemic GC muscles in WT and MɸKO mice on day 21 after HLI. Scale bars: 20 μm. n = 5–6 mice per group, unpaired, 2-tailed Student’s t test. The right panel shows quantification. (G) H&E staining of ischemic and non-ischemic adductor (AD) muscles in WT and MɸKO mice on day 7 after HLI. Arrowheads show collateral arteries. Scale bars: 50 μm. The right panels show quantification of diameter and wall thickness of collateral arteries (unpaired, 2-tailed Student’s t test). (H) H&E staining of ischemic and non-ischemic GC muscles in WT and MɸKO mice on day 3 (D3), D7, and D21 after HLI. Scale bars: 20 μm. The right panels show quantification of percentage of necrotic and regenerating myofiber in these muscles (2-way ANOVA followed by Bonferroni’s multiple-comparison test). Data are mean ± SEM. n = 4–6. *P < 0.05; **P < 0.01; ***P < 0.001.