Chronic Estrogen Treatment Promotes Human Microvascular Endothelial Dysfunction Through Sex-Specific Regulation of Ceramide Formation

Risk for cardiovascular disease (CVD) is elevated in individuals assigned female at birth and women of trans experience (assigned male at birth) undergoing long-term estrogen therapy (e.g. contraception, gender-affrmation). We have previously shown that chronic estrogen treatment induces microvascular endothelial dysfunction in human arterioles collected from those assigned male and female at birth. The mechanism via which estrogen causes microvascular damage remains unknown. Estrogen can increase expression of the ceramide-forming enzyme neutral sphingomyelinase (NSmase), and elevated ceramides promote cellular oxidative stress. Together, we hypothesize that estrogen induces microvascular endothelial dysfunction via NSmase-mediated ceramide formation. Human arterioles from otherwise healthy adults were treated with 17β-estradiol (E2; 100nM) +/- NSmase inhibitor (GW4869; 4μM, 24hrs). Vascular response to increasing flow rates was assessed using live videomicroscopy. Sex-specific human umbilical vein endothelial cells were treated with E2+/-GW4869 (48hrs), and H 2 O 2 levels were measured using the fluorescent indicator peroxy yellow 1 (PY1). Endothelial ceramide levels were measured using liquid chromatography, tandem mass spectrometry. Exposure to estrogen impaired dilation to flow in arterioles from those assigned male and female at birth, an effect that was prevented by inhibition of NSmase (%Mean Dilation±SE; E2 vs E2+GW4869; male: 11.20±4.99% vs 48.43±7.15%, n=3 both, p=0.037; female: 1.78±1.99% vs 40.35±8.46%, n=5 both, p=0.006; two-way ANOVA). Estrogen treatment increased endothelial H 2 O 2 production in cells from both sexes which was diminished by GW4869 treatment (%Change from control±SE; E2 vs E2+GW4869; male: 20.90±5.86% vs 2.85±1.99%, n=11 both, p=0.009*; female: 63.33±13.65%, n=10 vs 25.25±7.64%, n=9, p=0.031*, unpaired t-test*). Endothelial long-chain ceramide levels were higher in cells from biological males compared to females (pmol/nmol phosphate; male 5.90±2.19, n=4 vs female 2.18±0.86, n=5; p=0.009*). While estrogen treatment increased cellular ceramides in both sexes, ceramide accumulation was greater in cells from biological males compared to females (%change from baseline: 65.97±53.64% vs 6.08±7.86%, p=0.041*). Together, these results show that estrogen-induced microvascular endothelial dysfunction can be prevented through inhibition of NSmase-mediated ceramide formation. There is also notable sexual dimorphism in endothelial ceramide levels both at baseline and in response to estrogen. This study offers mechanistic insight into how chronic estrogen therapy may contribute to microvascular dysfunction and elevate the risk of CVD in patients. Furthermore, it presents the inhibition of NSmase as a plausible therapeutic approach to safeguard microvascular integrity during estrogen therapy. NHLBI R01HL160752 (J.K.F.), NHLBI K08HL141562 (J.K.F.), and AHA predoctoral fellowship grant 909315 (G.S.). This is the full abstract presented at the American Physiology Summit 2024 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.