Femoral artery ligation induces a lipid-associated macrophage program in ischemic skeletal muscle

Peripheral artery disease (PAD) reduces blood flow to the lower limbs, resulting in ischemia, injury, and inflammation of the lower limb muscles. The global cases of PAD are expected to increase by 220% by 2050, emphasizing the need to better understand the disease and identify new therapies to limit ischemic muscle damage. Macrophages play key roles in muscle damage, repair, and remodeling; however, the dominant macrophage phenotypes that populate the ischemic skeletal muscle in PAD remain incompletely defined. Lipid-associated macrophages (LAMs) are a macrophage phenotype characterized by enhanced lipid uptake, phagocytosis, and expression of lipid-handling and tissue-remodeling genes, including Spp1, Trem2, Gpnmb, and Lgals3. LAMs have been identified in metabolic and inflammatory diseases marked by lipid accumulation and tissue damage, such as obesity and steatotic liver disease, but whether a similar macrophage program arises in ischemic skeletal muscle during PAD has not been reported. Our study objective was to define the dominant immune cell populations and injury-associated programs that emerge in the ischemic skeletal muscle during PAD. We hypothesized that skeletal muscle ischemia triggers a hypoxia- and cell death-related signaling environment that promotes macrophage heterogeneity and preferential polarization toward a LAM state. Using femoral artery ligation (FAL), a well-established murine model of PAD, we combined bulk transcriptomics with histopathology, immunofluorescence, and flow cytometry to assess pathology and immune composition in the gastrocnemius muscle. Transcriptomic and histopathological analyses revealed that FAL induced a significantly upregulated hypoxic and necroptotic phenotype accompanied by prominent leukocyte infiltration. Transcriptomic analysis, flow cytometry, and immunohistochemistry revealed significant expansion of CD45 + cells, with macrophages (F4/80 + CD64 + and/or CD68 + ) constituting the dominant immune population in the ischemic muscle. These analyses further revealed a heterogeneous macrophage phenotype, including both pro-inflammatory and anti-inflammatory polarization states and the presence of monocyte-derived and resident macrophage subtypes. Strikingly, bulk RNA sequencing identified robust induction of canonical LAM genes ( Spp1, Trem2, Gpnmb, Lgals3, Fabp5, and Cd9) and differential expression of upstream regulators of LAM programs. In parallel, lipid metabolic pathways, lipid droplet accumulation, and fibro-adipogenic progenitor-associated genes linked to adipocyte deposition were increased, consistent with a lipid-rich microenvironment that supports a LAM phenotype. Overall, our findings demonstrate that PAD-induced skeletal muscle ischemia is defined by hypoxia, necroptosis, macrophage expansion and diversification dominated by a lipid-associated phenotype. Our results refine current understanding of macrophage identity in ischemic skeletal muscle and highlight LAM-related pathways as potential targets for modulating inflammation and remodeling in PAD Funding: National Heart, Lung, and Blood Institute (NHLBI): HL175929National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS): AR007612American Heart Association: AHA 970490 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.