Introduction
Cardiovascular diseases (CVD) show sex-specific differences in onset and severity. Premenopausal women have a lower incidence of CVD than age-matched men, a difference that diminishes after menopause. The functional properties of the surface of endothelial cells (ECs), forming the inner vascular layer, are crucial for the maintenance of vascular health. This includes the EC cortex, an actin-rich layer 50-150 nm beneath the plasma membrane, and the carbohydrate-rich endothelial glycocalyx (eGC). EC functions such as the release of vasoactive substances (e.g. nitric oxide, NO) and vascular permeability are closely linked to nanomechanical properties of the EC surface and their dynamic remodeling. ECs with a soft and upright eGC produce more NO and show less leukocyte adhesion. Deterioration of the eGC is a causal factor in the development of vascular inflammation. Since estradiol appears to have vasculoprotective effects, this study investigates how female sex hormones affect endothelial function.
Material and Methods
Human umbilical vein endothelial cells (HUVECs) were treated in vitro with estradiol, progesterone, the estradiol receptor (ER) α-selective antagonist MPP, or the ER β-selective antagonist PHTPP for 24 h. Nanomechanical properties (cortical stiffness, eGC stiffness and height) were analyzed using atomic force microscopy (AFM)-based indentation and validated by immunofluorescence staining. NO production was quantified in supernatants. GlycoCheck camera measurements were performed in healthy women (aged 20-30 years) at three phases of the menstrual cycle: early follicular, preovulatory and midluteal.
Results
Estradiol treatment induced dose-dependent changes in endothelial function. Low concentrations (0.05–0.1 nM, early follicular) improved eGC height and were associated with higher NO levels. In contrast, high estradiol concentrations (1–15 nM, preovulatory) damaged the eGC but did not alter NO levels compared to control. The ERβ antagonist PHTPP abolished the beneficial effect of low-dose estradiol. Low progesterone concentrations (1–10 nM, follicular + preovulatory) had no effect on eGC height, whereas high concentrations (50 nM, midluteal) improved it. In vivo, GlycoCheck measurements performed in healthy women showed a trend towards an increased perfused boundary region (PBR), indicating a damaged eGC during the preovulatory phase, coinciding with high estradiol levels.
Discussion
Varying physiological concentrations of estradiol and progesterone, as observed throughout the menstrual cycle, exert opposing effects on the nanomechanics of the endothelial surface — a key feature in flow-induced vasodilation and vascular homeostasis. While progesterone may support endothelial stability by increasing eGC height, estradiol tended to reduce eGC height and NO concentration, effects likely mediated through ERβ signaling. Since EC nanomechanics are central to vascular integrity and function, the demonstrated hormonal influence on these mechanical properties may provide a mechanistic explanation for how fluctuating and later declining female sex hormone levels contribute to the loss of vascular protection and increased cardiovascular risk after menopause.
A.H. was supported by DZHK doctoral scholarship.
