Single cell compendium of the muscle microenvironment in peripheral artery disease reveals capillary endothelial heterogeneity and activation of resident macrophages

Background Peripheral artery disease (PAD) is caused by atherosclerosis and chronic narrowing of lower limb arteries leading to decreased muscle perfusion and oxygenation. Current guidelines for treating PAD include endovascular strategies or bypass surgery but long-term outcomes have been suboptimal. This is likely due to our limited understanding of the contribution of the microvasculature as well as other cell types, in particular macrophages, to PAD skeletal muscle pathophysiology. We used single cell sequencing to investigate cellular and transcriptional heterogeneity of the skeletal muscle microenvironment in PAD. Methods Samples from the medial head of the gastrocnemius muscle of individuals undergoing either lower limb aneurysm surgery (controls) or PAD bypass surgery (PAD) were collected. Samples were either frozen for histological evaluation (control: n=4; PAD: n=6) or were immediately processed for single cell RNA sequencing of mononuclear cells (control: n=4; PAD: n= 4). Bioinformatic tools were used to annotate cell types and their subpopulations, to study transcriptional changes and to analyze cellular interactions. Results We generated a dataset comprised of 106,566 high-quality, deep-sequenced cells that compose the muscle microenvironment. Focusing on endothelial cells (ECs) and macrophages, we confirmed the presence of ATF3/4+ ECs with angiogenic and immune regulatory capacities in human muscle and found that their transcriptional profile profoundly alters during PAD. Also, capillary ECs display features of endothelial to mesenchymal transition. Furthermore, we identified LYVE1 hiMHCIIlow resident macrophages as the dominant macrophage population in human muscle, even under a chronic inflammatory condition such as PAD. During PAD, LYVE1 hiMHCIIlow macrophages get activated and acquire a more pro-inflammatory profile. Finally, we map strong intercellular communication in the muscle microenvironment, which is significantly altered in PAD. Conclusions The dataset we present here provides a highly valuable resource for gaining deeper insights into the critical roles that cells in the muscle microenvironment may play in PAD skeletal muscle pathology. We propose that targeting the crosstalk between ECs and macrophages could provide novel insights for developing effective treatments against this disease. ### Competing Interest Statement The authors have declared no competing interest. * PAD : peripheral artery disease IC : intermittent claudication CLI : critical limb ischemia ABI : ankle brachial index scRNAseq : single cell RNA sequencing EC : endothelial cell MoMac : monocytes/macrophages