Single-cell compendium of muscle microenvironment in peripheral artery disease reveals altered endothelial diversity and LYVE1+ macrophage activation.

Peripheral artery disease (PAD) results from atherosclerosis and chronic narrowing of lower limb arteries, leading to decreased muscle perfusion. Current treatments are suboptimal, partly due to limited understanding of PAD muscle pathology. Here we used single-cell RNA sequencing and spatial transcriptomics to analyze the composition of the muscle microenvironment in non-ischemic patients and patients with PAD. We identified ATF3/ATF4⁺ endothelial cells (ECs) that exhibit altered angiogenic and immune regulatory profiles during PAD and confirmed that ATF4 signaling in ECs is required for effective ischemia recovery. In addition, capillary ECs display features of endothelial-to-mesenchymal transition. Furthermore, LYVE1^hiMHCII^low macrophages are the dominant macrophage population in human muscle, adopting a more pro-inflammatory profile during PAD. Finally, we analyzed alterations in intercellular communication within the muscle microenvironment during PAD and confirmed that EC-derived factors can influence macrophage polarization. This dataset deeply characterizes the PAD muscle microenvironment and provides a resource for exploration of targeted therapies.