Sex and stimulus-specific differences in endothelial cell senescence under hormone-free conditions

Sex differences in cardiovascular disease are well documented, with females often considered hormonally protected. However, some differences persist even after menopause, indicating nonhormonal influences. Endothelial dysfunction is an early contributor to cardiovascular disease, with endothelial cell senescence playing a key role. Senescence, an irreversible cell cycle arrest, can be replicative or stress-induced. This study investigates whether sex differences in endothelial senescence exist independent of hormonal influence and vary by stimulus. Senescence was induced by replication or irradiation in female and male human umbilical vein endothelial cells (HUVECs) (up to n = 7 each) cultured under hormone-free conditions. Senescence-associated β-galactosidase (SA-β-Gal) staining; telomere length; quantitative real-time PCR (RT-qPCR) of p21, p14, p16; and crystal violet assays were used to assess senescence. Replicative senescence was analyzed across passages 1-20 and stress-induced senescence 5 days after irradiation. Female HUVECs had a significantly longer replicative lifespan than male cells (P = 0.0012) despite similar proliferation. Telomere attrition occurred faster in male cells (P = 0.0034), with earlier expression of senescence markers. In contrast, after irradiation, female cells exhibited stronger senescence responses, including increased SA-β-Gal staining and elevated p21, p14, and p16 levels. This study identifies sex differences in endothelial cell senescence under hormone-free conditions, pointing to intrinsic cellular factors. Although male cells exhibited earlier senescence under replicative stress, female cells were more vulnerable to stress-induced senescence. Together, these results highlight the importance of sex- and stimulus-specific mechanisms in vascular aging.NEW & NOTEWORTHY This study reveals intrinsic, hormone-independent sex differences in endothelial cell senescence. Female HUVECs exhibit delayed replicative but enhanced stress-induced senescence compared with male cells. These opposing responses highlight that sex-specific mechanisms in vascular aging depend on the type of senescence stimulus. By using hormone-free conditions, the study underscores the importance of intrinsic cellular factors in endothelial biology and suggests that sex should be considered when investigating and targeting vascular aging and disease.