6\u03b2-Hydroxytestosterone, a metabolite of testosterone generated by CYP1B1, contributes to vascular changes in angiotensin II-induced hypertension in male mice

Ajeeth K Pingili,Brett L Jennings,Kamalika Mukherjee,Frank J Gonzalez,Kafait U Malik,Wadah Akroush

doi:10.1186/s13293-019-0280-4

Abstract

BackgroundPreviously, we showed that 6β-hydroxytestosterone (6β-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to angiotensin II (Ang II)-induced hypertension in male mice. This study was conducted to test the hypothesis that 6β-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension.MethodsEight- to 10-week-old intact or castrated C57BL/6 J (Cyp1b1+/+ and Cyp1b1−/−) mice were anesthetized for implantation of a micro-osmotic pump which delivered Ang II (700 ng/kg/day) or saline for 14 days. Mice were injected with 6β-OHT (15 μg/g b.w every third day), flutamide (8 mg/kg every day), or its vehicle. Blood pressure was measured via tail-cuff. Vascular reactivity, endothelial-dependent and endothelial-independent vasodilation, media to lumen ratio, fibrosis by collagen deposition, and reactive oxygen species production by dihydroethidium staining were determined in the isolated thoracic aorta.ResultsThe response of thoracic aorta to phenylephrine and endothelin-1 was increased in Ang II-infused Cyp1b1+/+ mice compared to intact Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice; these effects of Ang II were restored by treatment with 6β-OHT. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation of the aorta to acetylcholine in Cyp1b1+/+ but not Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice. 6β-OHT did not alter Ang II-induced endothelial dysfunction in Cyp1b1+/+ mice but restored it in Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice. Ang II infusion increased media to lumen ratio and caused fibrosis and reactive oxygen species production in the aorta of Cyp1b1+/+ mice. These effects were minimized in the aorta of Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice and restored by treatment with 6β-OHT. Treatment with the androgen receptor antagonist flutamide reduced blood pressure and vascular hypertrophy in castrated Ang II-infused mice injected with 6β-OHT.Conclusions6β-OHT is required for the action of Ang II to increase vascular reactivity and cause endothelial dysfunction, hypertrophy, and increase in oxygen radical production. The effect of 6β-OHT in mediating Ang II-induced hypertension and associated hypertrophy is dependent on the androgen receptor. Therefore, CYP1B1 could serve as a novel target for the development of therapeutics to treat vascular changes in hypertensive males.

Highlights

Hypertension is the leading cause of cardiovascular diseases, renal dysfunction, and end-organ damage, and biological sex plays a significant role in the pathogenesis of hypertension and associated end-organ damage [1,2,3,4]
Cyp1b1 gene disruption or castration in Cyp1b1+/+ and Cyp1b1−/− mice reduced aortic responses to vasoconstrictor agents caused by angiotensin II (Ang II) infusion, which was restored by 6β-OHT Ang II-induced hypertension was associated with an increased constriction of the isolated thoracic aortic rings (55% from Cyp1b1+/+ vehicle-treated group) (Fig. 1a) to maximal concentration of phenylephrine (PE) and (83% from Cyp1b1+/+ vehicle-treated group) to maximal concentration of endothelin-1 (ET-1); these increases were attenuated by Cyp1b1 gene disruption (24% for PE, 52% for ET-1 compared to Cyp1b1+/+ vehicle-treated group), (2020) 11:4
Cyp1b1 gene disruption or castration of Cyp1b1+/+ mice reduced endothelial dysfunction caused by Ang II infusion, which was restored by 6β-OHT Ang II infusion caused endothelial dysfunction in the aorta, as determined by the effect of ACh to induce maximal relaxation of the aorta pre-constricted with PE (54% Cyp1b1+/+ vehicle-treated group) (Fig. 2a)

Summary

Introduction

Hypertension is the leading cause of cardiovascular diseases, renal dysfunction, and end-organ damage, and biological sex plays a significant role in the pathogenesis of hypertension and associated end-organ damage [1,2,3,4]. We demonstrated that the protective effect of 17-β estradiol against Ang II-induced hypertension and associated cardiovascular and renal pathophysiological changes are mediated most likely by its metabolite, 2-methoxyestradiol generated by CYP1B1 in female mice [11,12,13]. Since Ang II causes vascular dysfunction, hypertrophy, fibrosis, and reactive oxygen species production (ROS) [18], we hypothesized that 6βOHT mediates these vascular effects of Ang II in male mice To test this hypothesis, we investigated the contribution of 6β-OHT to the effects of Ang II to increase vascular reactivity, endothelial dysfunction, hypertrophy, fibrosis, and ROS production in Ang II-induced hypertension in the thoracic aorta of castrated Cyp1b1+/+ and Cyp1b1−/− mice that lack endogenous testosterone and 6β-OHT. This study was conducted to test the hypothesis that 6β-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension

Methods

Results

Conclusion