Collagen-targeted extracellular vesicles from exercised progenitors restore cardiac microcirculation via endothelial AMPK-eNOS-NO axis reactivation

Endothelial dysfunction, driven by impairment of the AMPK-eNOS-NO signaling axis, is a primary defect in diabetic cardiomyopathy (DCM). Extracellular vesicles (EVs) are key mediators of intercellular communication. EVs from exercised conditions possess enhanced bioactivity, but their therapeutic potential is limited by inefficient delivery. How to engineer EVs for targeted signaling restoration in the diseased cardiac endothelium remains unexplored. EVs were harvested from endothelial progenitor cells of treadmill-trained mice (Ex-EVs). We engineered a bifunctional peptide, CP05-SILY, where CP05 anchors to EV surface protein CD63 and SILY binds collagen, to create collagen-targeting EVs (Ex-EV-CS). Their binding, uptake, and functional effects were assessed in cardiac endothelial cells under metabolic stress and in a DCM mouse model. Mechanistic insights were gained through transcriptomic/metabolomic analysis, pharmacological inhibition (AMPK, eNOS) NO scavenger, and endothelial-specific Ampkα1 knockdown. Candidate protein cargoes were further examined to explore cargo-related contributions. Ex-EV-CS showed superior binding to glycated collagen and significantly enhanced retention within the diabetic heart compared to plain Ex-EVs. This improved delivery translated to potent reactivation of the AMPK-eNOS-NO axis in endothelial cells, increasing nitric oxide bioavailability, reducing oxidative stress, and restoring endothelial migration, angiogenesis, and barrier integrity. In DCM mice, Ex-EV-CS treatment effectively rescued myocardial perfusion, promoted mature microvascular formation, and reduced fibrosis. The therapeutic benefits were abolished upon disruption of AMPK or eNOS signaling, or by endothelial Ampkα1 deletion. Control EVs lacking either the CP05 anchor or SILY targeting motif were ineffective, confirming the necessity of the engineered targeting module. Candidate protein cargo analysis showed enrichment of endothelial repair-related proteins in Ex -EVs. We engineered EVs to achieve targeted intercellular communication, specifically delivering exercise-induced pro-signaling cargo to diseased endothelial cells. This strategy successfully restores the critical AMPK-eNOS-NO signaling pathway, rebuilds endothelial-centric intercellular communication networks, and rescues microvascular function in DCM, highlighting a novel signaling-based therapeutic paradigm.