Elevated Estrogen Induces Senescence in Male Endothelial Cells Via Neutral Sphingomyelinase-Mediated Ceramide Formation
Authors: SenthilKumar Gopika , Shult Carolyn , Bordas-Murphy Henry , Freed Julie 
Published: May 2023

Prevalence of cardiovascular disease is higher among trans-females undergoing estrogen therapy compared to cis-females and males. Our previous work has shown that chronic exposure to elevated estrogen induces microvascular endothelial dysfunction in arterioles collected from healthy biological males, however the mechanism contributing to dysfunction is unknown. Conflicting evidence has shown that estrogen can both promote or prevent cell senescence, a phenotypic change characterized by replicative arrest and endothelial dysfunction. Since estrogen is capable of activating neutral sphingomyelinase (NSmase), an enzyme that increases cellular levels of the pro-senescent sphingolipid ceramide, we hypothesized that elevated estrogen induces endothelial cell senescence in biological males through the activation of NSmase. To study this, primary male human umbilical vein endothelial cells (HUVECs) were treated with increasing doses of 17-beta estradiol (E2; 0.5nM to 100nM) for 24hrs with or without a neutral sphingomyelinase inhibitor (GW4869 5μM). Cells were also exposed to C2-ceramide (5μM) or inactive dihidro-C2-ceramide (5μM) and maintained for 48hrs in 1% serum EBM-2 media prior to beta-galactosidase staining (biomarker of senescence). Treatment groups were imaged using 20x bright-field microscopy. Total number of positive beta-galactosidase-stained cells were counted and normalized to total number of cells per image field (mean%±SEM reported; n=3 patients for all); counters were blinded to treatment group. One-Way ANOVA with alpha<0.05 was used to compare between groups. Male HUVECs exhibited no change in percent positive cells with 0.5nM E2 treatment (25±11.4% vs 21.5±2.4% vehicle control), however had significantly higher percentage of positive cells when exposed to higher doses of E2 (1nM, 45.±11.5%; 50nM, 48.1±6.1%; 100nM, 45.5±2.6%; p<0.05 all). Treatment with ceramide (40.44±7.8% vs 11.2±4.3% control, p<0.05) but not dihydroceramide (14.89±12.1%) increased percentage of positive cells. Inhibition of neutral sphingomyelinase alone did not influence percent positivity (12.4±2.7% vs 11.2±4.3% control), however in comparison to 100nM E2 treatment, those treated with GW4869 and 100nM E2 exhibited a lower percentage of positive cells (45.5±2.6% 100nM E2 vs 19.22±7.0% E2+GW4869, p<0.05). These results suggest that elevated estrogen may cause an increase in endothelial cell senescence among biological males through the upregulation of neutral sphingomyelinase-mediated ceramide formation. Our work adds mechanistic insight by which estrogen may promote endothelial dysfunction among trans-females undergoing estrogen therapy and proposes the inhibition of NSmase-mediated ceramide formation as a novel therapeutic avenue for reducing CVD risk associated with gender transition. This work is supported by National Institutes of Health (NHLBI) K08 HL141562 (JKF) and American Heart Association Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.