Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study

Nebivolol (NEB) is a [beta]1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor.

Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17[beta]-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor N[omega]-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or N[omega]-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ER[alpha] and ER[beta] total RNA was performed by semiquantitative PCR.

Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 +/- 0.03 pN/nm, E2: 0.87 +/- 0.03 pN/nm, vehicle: 2.19 +/- 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 +/- 0.04 pN/nm, all n = 60, P < 0.01). The decrease in stiffness occurred as soon as 5 min after starting NEB incubation. The effects are mediated through nongenomic ER[beta] pathways, as ER[alpha] is not translated into measurable protein levels in EAHy-926. Furthermore, NEB increased cell volume by 48 +/- 4% and apical surface by 34 +/- 3%. E2 had comparable effects. Tamoxifen, ICI and N[omega]-nitro-L-arginine methyl ester substantially diminished the effects of NEB and E2.

NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ER[beta] pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.