Hypoxia conditioned adipose-derived stem cell-derived extracellular vesicle therapy improves cardiac function in a rat model of ischemic cardiomyopathy

Extracellular vesicles (EVs) are potential cell-free therapies for cardiac regeneration. Although adipose-derived stem cells (ADSCs) are easily obtained using minimally invasive procedures, therapeutic effects of hypoxically conditioned ADSC-derived EVs on the heart remain unknown. We aimed to verify whether hypoxic preconditioning enhances the therapeutic efficacy of ADSC-derived EVs. Lewis female rats were used to isolate ADSCs and establish an ICM rat model. ADSCs were cultured in a 48-h exosome-free medium under 5% or 20% O 2 , and EVs were recovered using polyethylene glycol and ultracentrifugation. An MI model was created by ligating the left anterior descending artery via mini thoracotomy; EF<45% was confirmed 2 weeks later. Rats were randomly assigned to three groups (PBS, normoxic EV, and hypoxic EV groups), each undergoing mini thoracotomy and myocardial injection. Echocardiography was performed at 2- and 4-weeks post-administration to evaluate cardiac function, and histological analysis of fibrosis and angiogenesis was performed at 4 weeks. We performed miRNA sequencing on both EVs and RNA sequencing on the myocardium 2 days after administration. Functional analysis of EV microRNAs suggested that hypoxia increased levels of miRNAs involved in suppressing fibrosis. Echocardiography at 2 and 4 weeks post-EV administration showed greater improvement in cardiac function (reduction in diastolic and systolic diameters, and improvement in left ventricular EF) in the hypoxic ADSC-derived EV group. Fibrosis reduced and vascularization increased in the hypoxic ADSC-derived EV group tissues. On day 2 after hypoxic ADSC-derived EV administration, RNA sequencing of myocardial infarction border zones showed increased expression of multiple genes associated with antifibrotic, anti-inflammatory, and angiogenic processes in the heart. GSEA identified increased expression of acute inflammatory responses. Hypoxic ADSC-derived EV administration induces acute inflammation and suppresses late-stage fibrosis by modulating immunity, thereby contributing to improved cardiac function. They may be an effective option for cardiac regenerative therapy.