Extracellular vesicles and their miRNA cargo in response to physical exercise: potential involvement in oxidative stress regulation – Insights from the HEBE Study

Introduction: Physical exercise (PE) is crucial for maintaining health across all ages, with regular PE playing a key role in preventing non-communicable diseases. In particular, moderate-intensity PE has been shown to reduce inflammation and oxidative stress, two highly interconnected processes. Both acute and chronic PE stimulate various tissues and organs to release extracellular vesicles (EVs), which allow cell-to-cell long-distance communication, contributing to whole-body homeostasis. EVs carry bioactive molecules, including microRNAs (miRNAs), which are post-transcriptional regulators of gene expression influenced by external factors like PE, and are promising biomarkers of effect. This study aims to investigate the role of EVs and their miRNA content as potential mediators of the relationship between PE, oxidative stress, and inflammation. Methods: After signing informed consent, 91 participants from the HEBE project (Healthy aging versus inflamm-aging: the role of physical Exercise in modulating the Biomarkers of age-associated and Environmentally determined chronic diseases) were enrolled. Clinical parameters were assessed, and blood samples were collected for circulating marker measurements before (T0) and after (T1) a 6-month personalized PE intervention. Plasma-derived EVs were isolated by ultracentrifugation and characterized for size distribution and concentration using Nanoparticle Tracking Analysis (NTA). After EV-miRNAs extraction, the miRNome (755 miRNAs) was profiled using OpenArray technology. EV size distribution and total concentration were compared between T0 and T1. Differentially expressed miRNAs between T0 and T1 were identified through statistical analysis and evaluated by Gene Ontology (GO) enrichment analysis. Results: No significant difference in EV size distribution was observed between T0 and T1. Following the PE intervention, five EV-associated miRNAs were upregulated (hsa-miR-483-5p, hsa-miR-181a, hsa-miR-376c, hsa-miR-376a, hsa-miR-222), while two were downregulated (hsa-miR-98, hsa-miR-664). Enrichment analysis revealed a significant involvement of key cellular pathways, including apoptosis and oxidative stress response. Conclusions: Our findings suggest that EVs and their miRNA content play a role in the regulation of key biological pathways, including the cellular response to oxidative stress, in response to PE. This study provides new insights into the molecular mechanisms underlying the beneficial effects of PE, highlighting the potential role of EV-miRNAs as mediators of PE-induced adaptations. Further research is needed to fully elucidate their functional impact.