Multi-omics integrative approach of senescent endothelial cells and derived extracellular vesicles in a replicative senescence model [miRNA-seq]
Aging is a major unmodifiable risk factor for cardiovascular disease (CVD). Replicative endothelial senescence (RES), characterized by permanent cell-cycle arrest and a senescence-associated secretory phenotype (SASP), is a hallmark of vascular aging and contributes to endothelial dysfunction. However, RES molecular mechanisms, particularly the role of extracellular vesicles (EVs), remain poorly understood. To elucidate these mechanisms, an integrated multi-omics approach (proteomics, transcriptomics, and miRNA profiling) was used to characterize senescent endothelial cells (ECs) and their secreted EVs compared with early-passage counterparts. Senescent ECs and EVs displayed canonical senescence signatures, exhibiting enrichment of cell cycle, DNA damage response, p53 and NF-?B signaling, and SASP components, which are strongly linked to CVD.  Senescent EVs also showed a marked suppression of RNA metabolism, chromatin organization, and repair machinery, while carrying pro-inflammatory, adipogenic, and angiogenic mediators, reinforcing their role as vectors of secondary cellular senescence. Multi-omics integration revealed a coordinated regulatory network involving transcription factors (e.g., REST) and miRNAs. Angiogenic regulators miR-22-3p and miR-126-5p also emerged as RES mediators in both ECs and EVs, with miR-22-3p exhibiting compartment-specific regulation. Downregulation of key miRNAs (miR-335-3p, miR-590-3p, miR-29, and miR-17 families) promoted the derepression of downstream targets, driving endothelial phenotypic drift and dysfunction, primarily characterized by impaired PI3K–Akt signaling, maladaptive angiogenesis, and extracellular matrix remodeling. Our study provides a comprehensive map of RES-driven molecular alterations, identifying potential candidate biomarkers and therapeutic targets for age-related CVD. These findings also emphasize the role of EVs in propagating senescence and CVD risk, opening perspectives for EV-based interventions to counteract vascular aging. Overall design: miRNA profiling of senescent and early-passage HUVECs and their respective secreted extracellular vesicles. Cells studied within passages 2–8 (early passage; PD <20) were regarded as early-passage endothelial cells (ECs), whereas those passaged 28–38 times (PD >96) were regarded as senescent ECs.