Multi-omics integrative approach of senescent endothelial cells and derived extracellular vesicles in a replicative senescence model

Abstract

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. However, the molecular mechanisms underlying RES, particularly the role of extracellular vesicles (EVs), remain poorly understood. To this aim, an integrated multi-omics approach (proteomics, mRNA, and miRNA profiling) was applied to characterize senescent and early-passage human umbilical vein endothelial cells (HUVECs) and their secreted EVs. Senescent HUVECs and EVs displayed a canonical senescence profile, including cell-cycle arrest, DDR activation, NF-{kappa}B signaling, and SASP induction, alongside marked suppression of RNA metabolism, ribosome biogenesis, and DNA repair. Multi-omics integration has linked endothelial senescence to vascular aging, maladaptive angiogenesis, and ECM remodeling, which are key processes in CVD development. Moreover, senescent EVs propagate senescence by exporting fewer reparative and antioxidant factors while carrying pro-fibrotic, adipogenic, and angiogenic mediators. Multi-omics profiling revealed consistent transcript-protein changes in senescent cells and EVs, defining a robust molecular signature of RES, including endothelial cell-specific markers and post-transcriptional regulators (lncRNAs and miRNAs). Among these, miR-22-3p and miR-126-5p emerged as key modulators in both cells and EVs, with miR-22-3p demonstrating compartment-specific regulation. Integration further uncovered a coordinated regulatory network involving miRNAs (miR-23, miR-335-3p, miR-29 family, miR-590-3p, and miR-126-5p) that were inversely correlated with transcription factors (e.g., REST, KLFs, and ZNFs) and downstream targets, collectively driving endothelial senescence through impaired PI3K-Akt signaling. Although further validation is needed, these findings provide new insights into the molecular mechanisms of RES and open perspectives for modulating secondary senescence and age-related vascular diseases.