Addition of Estradiol to Cross-Sex Testosterone Therapy Reduces Atherosclerosis Plaque Formation in Female ApoE-/- Mice

Abstract

The contributions of estradiol and testosterone to atherosclerotic lesion progression are not entirely understood. Cross-sex hormone therapy (XHT) for transgender individuals dramatically alters estrogen and testosterone levels and consequently could have widespread consequences for cardiovascular health. Yet, no preclinical research has assessed atherosclerosis risk after XHT. We examined the effects of testosterone XHT after ovariectomy on atherosclerosis plaque formation in female mice and evaluated whether adding low-dose estradiol to cross-sex testosterone treatments after ovariectomy reduced lesion formation. Six-week-old female ApoE-/- C57BL/6 mice underwent ovariectomy and began treatments with testosterone, estradiol, testosterone with low-dose estradiol, or vehicle alone until euthanized at 23 weeks of age. Atherosclerosis lesion progression was measured by Oil Red O stain and confirmed histologically. We found reduced atherosclerosis in the estradiol- and combined testosterone/estradiol-treated mice compared with those treated with testosterone or vehicle only in the whole aorta (-75%), aortic arch (-80%), and thoracic aorta (-80%). Plaque size was similarly reduced in the aortic sinus. These reductions in lesion size after combined testosterone/estradiol treatment were comparable to those obtained with estrogen alone. Testosterone/estradiol combined therapy resulted in less atherosclerosis plaque formation than either vehicle or testosterone alone after ovariectomy. Testosterone/estradiol therapy was comparable to estradiol replacement alone, whereas mice treated with testosterone only fared no better than untreated controls after ovariectomy. Adding low-dose estrogen to cross-sex testosterone therapy after oophorectomy could improve cardiovascular outcomes for transgender patients. Additionally, these results contribute to understanding of the effects of estrogen and testosterone on atherosclerosis progression.

Figure 1.

Quantification of aorta ORO stain. Percent stained area is shown for (A) whole aorta, (B) aortic arch, and (C) thoracic aorta (vehicle control: n = 5; T, E, and T+E: n = 6 per group). Data are presented as mean ± SEM. There was a significant relationship between treatment and ORO staining for the whole aorta (F = 9.07, P = 0.006), aortic arch (F = 7.36, P = 0.002), and thoracic aorta (F = 7.67, P = 0.0015). Post hoc Tukey-corrected tests demonstrated that the E and T+E groups had significantly less lesion formation than mice treated with vehicle or with T alone, for whole aorta, aortic arch, and thoracic aorta. No other comparisons were significantly different. Significance compared with vehicle controls: *P = 0.03, **P = 0.02. Significance compared with T: ^#P = 0.03, ^##P = 0.02, ^###P < 0.01. (D) A representative aorta for each group with red quantification on ImageJ is shown.

Figure 2.

H&E staining of the aortic root. Immunohistochemistry images are shown for representative mouse groups treated with vehicle control (C), T, E, and combined T+E after OVX. Photographs were taken at ×4 magnification; black arrows indicate large plaque formation.

Figure 3.

Average lesion area in H&E-stained aortic root. Average lesion area is presented as fold change compared with C, with bars representing SEM. We found a significant relationship between average lesion area per H&E-stained aortic root sample and treatment (F = 10.92, P = 0.0002). Post hoc Tukey-corrected tests demonstrated a reduction in average lesion area for E-treated and T+E-treated mice, compared with both C- and T-treated mice. Significance compared with C, *P < 0.01. Significance compared with T, ^#P < 0.01.