Dihydrotestosterone Induces Arterial Stiffening in Female Mice

Abstract

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.

Ovariectomy-Induced Arterial Stiffening Differs From Vascular Aging and Is Reversed by GPER Activation

“…The majority of values in intact mice were below the lower limit of detection of 2 pg/mL, as previously reported. 35,38 Including these values at the lower limit, we found that average estradiol levels were 13.2±21.7 pg/ mL in intact 20-week-old mice and 2.6±1.1 pg/mL in intact 52-week-old mice (n=7 per group). Both ovariectomy and aging increased intracarotid PWV and aortic PWV to a similar extent (Figure 1A and 1B).…”

“…Verhoeff–Van Gieson stain was used to quantify elastin, Masson trichrome stain was used for collagen and smooth muscle, and Alcian blue stain was used for glycosaminoglycans, as previously described. 20,28,35 Analysis was performed using Adobe Photoshop (v.24.5.0). For each tissue section, the entire cross section was imaged, and the number of pixels stained with the selected color was counted and expressed as an area fraction (percent of pixels with positive staining within the tissue area).…”