Abstract Body: Background: Peripheral artery disease (PAD) affects >230M people worldwide and is a leading cause of limb pain, non-healing wounds, and, in severe cases, limb loss. Diabetes markedly increases PAD risk, yet the molecular mechanisms driving this vulnerability remain poorly defined. We hypothesized that diabetes disrupts cellular interactions within the vascular niche, impairing tissue repair in PAD.

Aim: To characterize the cellular landscape and identify transcriptomic and cell-signaling mechanisms underlying diabetes-associated PAD.

Methods: Skeletal muscle tissues were collected from PAD patients undergoing amputation including non-ischemic (tibialis anterior) and ischemic (flexus hallucis longus) muscles, for histology and qPCR. To model diabetes-associated PAD, hindlimb ischemia was performed in streptozotocin-induced 18w diabetic and non-diabetic Apoe^-/- mice, with gastrocnemius harvested 2w later from ischemic and non-ischemic limbs for single-cell RNA sequencing (scRNA-seq), histology and qPCR.

Results: In amputation tissues, ischemic muscles showed reduced microvessel density with increased collagen IV, and microvessels stained for endothelial cells (ECs), fibro-adipo-progenitors (FAPs), and perivascular cells. In mice, scRNA-seq identified 24 mononuclear populations, with FAPs and ECs as the largest fractions. FAP numbers increased with ischemia, but this enrichment was lost in diabetes, whereas EC numbers increased regardless of diabetes. In diabetic ischemic FAPs, gene ontology revealed positive enrichment for leukocyte adhesion and cell activation, and negative enrichment for extracellular matrix and cell projection organization. Cell-cell communication modeling identified FAPs and ECs as key signaling hubs, with signaling strength reduced in diabetes. Pathway analysis of FAP–EC interactions revealed increased thrombospondin signaling in ischemia, further elevated under diabetic conditions.

Conclusion: Diabetes disrupts FAP–EC communication via increased thrombospondin signaling, highlighting thrombospondins as novel contributors to PAD pathogenesis and potential therapeutic targets. Further work is needed to determine whether targeting these pathways can translate into improved outcomes for patients with diabetes-associated PAD.