Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and endothelial dysfunction. We hypothesized that sex differences exist in vascular aging processes and would be attenuated by global deletion of the G protein-coupled estrogen receptor. Blood pressure was measured by tail cuff plethysmography, pulse wave velocity (PWV) and echocardiography were assessed with high resolution ultrasound, and small vessel reactivity was measured using wire myography in adult (25 weeks) and middle-aged (57 weeks) male and female mice. Adult female mice displayed lower blood pressure and PWV, but this sex difference was absent in middle-aged mice. Aging significantly increased PWV but not blood pressure in both sexes. Adult female carotids were more distensible than males, but this sex difference was lost during aging. Acetylcholine-induced relaxation was greater in female than male mice at both ages, and only males showed aging-induced changes in cardiac hypertrophy and function. GPER deletion removed the sex difference in PWV as well as ex vivo stiffness in adult mice. The sex difference in blood pressure was absent in KO mice and was associated with endothelial dysfunction in females. These findings indicate that the impact of aging on arterial stiffening and endothelial function is not the same in male and female mice. Moreover, nongenomic estrogen signaling through GPER impacted vascular phenotype differently in male and female mice. Delineating sex differences in vascular changes during healthy aging is an important first step in improving early detection and sex-specific treatments in our aging population.

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Plasma soluble prorenin receptor (sPRR) displays sexual dimorphism and is higher in women with type 2 diabetes mellitus (T2DM). However, the contribution of plasma sPRR to the development of vascular complications in T2DM remains unclear. We investigated if plasma sPRR contributes to sex differences in the activation of the systemic renin-angiotensin-aldosterone system (RAAS) and vascular damage in a model of high-fat diet (HFD)-induced T2DM. Male and female C57BL/6J mice were fed either a normal fat diet (NFD) or an HFD for 28 weeks to assess changes in blood pressure, cardiometabolic phenotype, plasma prorenin/renin, sPRR, and Ang II. After completing dietary protocols, tissues were collected from males to assess vascular reactivity and aortic reactive oxygen species (ROS). A cohort of male mice was used to determine the direct contribution of increased systemic sPRR by infusion. To investigate the role of ovarian hormones, ovariectomy (OVX) was performed at 32 weeks in females fed either a NFD or HFD. Significant sex differences were found after 28 weeks of HFD, where only males developed T2DM and increased plasma prorenin/renin, sPRR, and Ang II. T2DM in males was accompanied by non-dipping hypertension, carotid artery material stiffening, and aortic ROS. sPRR infusion in males induced vascular thickening instead of material stiffening caused by HFD-induced T2DM. While intact females were less prone to T2DM, OVX increased plasma prorenin/renin, sPRR, and SBP. These data suggest that sPRR is a novel indicator of systemic RAAS activation and reflects the onset of vascular complications during T2DM regulated by sex.

Background: Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome. These patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via regulation of GPER. Methods: The impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n=8-9/group). Results: GPER mRNA was only decreased by DHT (P=0.001), while ERα expression was significantly suppressed by all hormones (P<0.0001). While blood pressure was not different between groups (P= 0.59), there was a dose-dependent increase in body weight (control 22±2 g, single dose 24±2 g, double dose 26±2 g; P=0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9±0.3 m/s, single dose 4.3±0.8 m/s, double dose 4.8±1.0 m/s). Histological analysis of aortic sections using Masson’s trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P=0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20±5, single dose 10.5 ± 5.6, double dose 10±4 copies/ng; P=0.001) and ERα (control 54±2, single dose 24±13, and double dose 23 ± 12 copies/ng; P=0.003). Conclusions: These findings indicate that testosterone promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important not only for polycystic ovarian syndrome patients but also women using testosterone for fitness, gender transitioning, or reduced libido.

Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome (PCOS). PCOS patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Our previous work showed significant downregulation of the G protein‐coupled estrogen receptor (GPER) as well as the nuclear estrogen receptor ERα in cultured vascular smooth muscle cells in response to the testosterone metabolite dihydrotestosterone (DHT), which cannot be aromatized to estrogen. Therefore, we hypothesized that DHT may induce arterial stiffness in female mice via downregulation of vascular estrogen receptors. Two groups of female wildtype mice were treated with DHT via silastic capsule for 4 weeks; one group received a dose designed to mimic DHT levels in men while the other group received twice this dose. While post‐treatment systolic blood pressure was not different between the control, single dose, and double dose groups (111 ± 10, 106 ± 3, 110 ± 7, n=8 per group; P=0.22), intracarotid vascular stiffness measured via pulse wave velocity showed a more than two‐fold increase in single dose (4.3 ± 0.8 m/s) and double dose (4.8 ± 1.0 m/s) DHT treated mice when compared with non‐treated controls (1.9 ± 0.3 m/s; P<0.0001). These findings suggest that high testosterone levels in females may promote arterial stiffening and cardiovascular damage independent of changes in blood pressure. Future studies will include histological analysis of aortic sections using Masson’s trichrome to assess changes in the extracellular matrix as well as droplet digital polymerase chain reaction to assess changes in estrogen receptor expression. In conclusion, these findings of arterial stiffening in response to increased DHT levels in females are important for not only PCOS patients but also women using testosterone for fitness, gender transitioning, or reduced libido.