In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle
Authors: Bedell Bradley R. , Kehmeier Mackenzie N. , Khurana Aleena , Henson Grant D. , Walker Ashley E. 
Published: May 2022

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p>0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p>0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p>0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.