Abstract 4372587: Plasma-Primed Pericyte-Derived Extracellular Vesicles Enhance Angiogenic Responses and Promote Recovery in Ischemic Limb Tissue

Introduction: Peripheral artery disease (PAD) is marked by reduced limb blood flow due to atherosclerosis, often leading to ischemia, non-healing wounds, or limb loss. Pericytes are critical for vascular homeostasis and angiogenesis via their secreted extracellular vesicles (EVs). However, under PAD-associated oxidative stress, pericyte-derived EVs (PDEVs) lose function. Cold atmospheric plasma (CAP), a non-thermal ionized gas composed of reactive oxygen and nitrogen species (RONS), can modulate cell signaling without cytotoxicity at low doses. Our study explores enhancing PDEVs’ angiogenic potential through low-dose CAP stimulation. Hypothesis: Priming healthy mouse skeletal muscle pericytes with CAP will yield PDEVs with enhanced angiogenic and regenerative activity. We hypothesize that CAP-primed PDEVs will improve endothelial cell function in vitro and promote blood flow recovery in vivo , offering a safe, acellular therapy for PAD. Methods: Primary pericytes were treated with CAP (50:50 argon:nitrogen) for durations of 0–120 seconds. After 24 hours, media were collected and EVs isolated by ultracentrifugation. EV identity was confirmed via nanoparticle tracking analysis and EV marker arrays. Proteomic changes were analyzed using LC-MS/MS, followed by bioinformatic enrichment of angiogenesis, migration, and inflammation-related pathways. For in vitro studies, HUVECs were treated with control or CAP-primed PDEVs and assessed for migration (scratch assay) and tubule formation (Matrigel). In vivo , unilateral hindlimb ischemia was induced in C57BL/6J mice by femoral artery ligation. PDEVs were injected intramuscularly on day 1. Blood flow recovery was tracked via PET imaging and laser speckle contrast imaging. Results: CAP exposure induced dose-dependent cytotoxicity in pericytes (p<0.05). While 5–15 sec CAP caused minimal cell death (~20% apoptotic vs. 10% in controls), 120 sec led to ~60% apoptosis. Thus, 5 sec CAP was selected for EV priming. CAP-primed PDEVs significantly enhanced HUVEC proliferation, migration, and tubule formation under hypoxia (p<0.05). In vivo , treated mice showed significantly faster blood flow recovery compared to controls (p<0.05). Histological analysis is ongoing. Conclusion: Low-dose CAP enhances the regenerative function of PDEVs by promoting angiogenic protein expression without substantial toxicity. This strategy offers a promising acellular therapy for PAD and other ischemic disorders with strong translational potential.