Generation and Validation of a Lower Limb Muscle Single-Cell RNA Sequencing Data Set Identifies Pathogenic Endothelial Metabolism in Peripheral Arterial Disease

Noral, M.; Alhusban, S.; Han, Q.; Pressley, A. H.; O'Keefe, S. H.; Leanhart, S.; Southerland, K. W.; McClung, J. M.; Annex, B. H.

doi:10.1101/2025.09.23.678171

bioRxiv · 10.1101/2025.09.23.678171

Generation and Validation of a Lower Limb Muscle Single-Cell RNA Sequencing Data Set Identifies Pathogenic Endothelial Metabolism in Peripheral Arterial Disease

Noral, M.·Alhusban, S.·Han, Q.·Pressley, A. H.·O'Keefe, S. H.·Leanhart, S.·Southerland, K. W.·McClung, J. M.·Annex, B. H.

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Abstract

BackgroundPeripheral arterial disease (PAD) results from atherosclerotic occlusion(s) in leg arteries. Chronic limb-threatening ischemia (CLTI) is the most severe form of PAD. Patients with CLTI suffer from rest pain, ulcers, or gangrene. Clinical outcomes remain poor in patients with CLTI and many investigational approaches, such as promoting angiogenesis, have failed. Understanding cell-specific vs. bulk-RNA changes within muscle offers an opportunity to better understand this disease. ObjectiveTo assess cell-specific alterations in gene and metabolism pathways in endothelial and muscle cells from leg muscle between patients with PAD vs. non-PAD controls. MethodsWe analyzed publicly available bulk RNA-seq leg muscle data from non-PAD controls, patients with intermittent claudication (IC) and CLTI. An emphasis was on glycolysis and the hexosamine biosynthetic pathway (HBP). Two single-cell RNA-seq datasets were integrated, cell types identified, and metabolism pathway scores calculated across identified cell types. Validations included quantitative mRNA in flow-sorted ECs, and immunofluorescence from clinically-phenotyped human samples. ResultsBulk RNA-seq revealed downregulation of both glycolysis and HBP in CLTI vs. IC or control tissues. Single-cell analysis uncovered cell-type specific changes. In patients with CLTI, gene expression in ECs showed increased glycolysis and decreased HBP. In myonuclei gene expression of both pathways were reduced. Flow-sorted ECs confirmed higher glycolysis and reduced HBP in CLTI vs. controls. Immunofluorescence analysis of muscle revealed significantly lower HBPECs (9.5% vs. 31.3%, n=5/group, p=0.004) and increased glycolytic ECs (42.0% vs. 14.4%, n=7/group, p<0.001) in CLTI vs. control. The findings were independent of capillary density. In IC patients, supervised exercise vs. optimal medical care reduced ischemic muscle PFKFB3+ ECs (2.67% vs. 14.61%, n=11 vs. 9, p<0.001). ConclusionsWe generated and validated a single-cell RNA-seq data set that confirms maladaptive metabolic reprogramming in CLTI ECs. Strategies designed to alter metabolism need to account for differences between bulk vs. single-cell analyses.

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Noral, M., Alhusban, S., Han, Q., Pressley, A. H., O'Keefe, S. H., Leanhart, S., Southerland, K. W., McClung, J. M., Annex, B. H.. 2025-09-25. Generation and Validation of a Lower Limb Muscle Single-Cell RNA Sequencing Data Set Identifies Pathogenic Endothelial Metabolism in Peripheral Arterial Disease. https://doi.org/10.1101/2025.09.23.678171

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