Supraphysiological Levels of Testosterone Induce Vascular Dysfunction via Activation of the NLRP3 Inflammasome

Abstract

Background: Both supraphysiological and subphysiological testosterone levels are associated with increased cardiovascular risk. Testosterone consumption at supraphysiological doses has been linked to increased blood pressure, left ventricular hypertrophy, vascular dysfunction, and increased levels of inflammatory markers. Activation of the NLRP3 inflammasome contributes to the production of proinflammatory cytokines, leading to cardiovascular dysfunction. We hypothesized that supraphysiological levels of testosterone, via generation of mitochondrial reactive oxygen species (mROS), activates the NLRP3 inflammasome and promotes vascular dysfunction. Methods: Male, 12 week-old C57Bl/6J (WT) and NLRP3 knockout (NLRP3^-/-) mice were used. Mice were treated with testosterone propionate [TP (10 mg/kg) in vivo] or vehicle for 30 days. In addition, vessels were incubated with testosterone [Testo (10^-6 M, 2 h) in vitro]. Testosterone levels, blood pressure, vascular function (thoracic aortic rings), pro-caspase-1/caspase-1 and interleukin-1β (IL-1β) expression, and generation of reactive oxygen species were determined. Results: Testosterone increased contractile responses and reduced endothelium-dependent vasodilation, both in vivo and in vitro. These effects were not observed in arteries from NLRP3^-/- mice. Aortas of TP-treated WT mice (in vivo), as well as aortas from WT mice incubated with testo (in vitro), exhibited increased mROS levels and increased caspase-1 and IL-1β expression. These effects were not observed in arteries from NLRP3^-/- mice. Flutamide [Flu, 10^-5 M, androgen receptor (AR) antagonist], carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 10^-6 M, mitochondrial uncoupler) and MCC950 (MCC950, 10^-6 M, a NLRP3 receptor inhibitor) prevented testosterone-induced mROS generation. Conclusion: Supraphysiological levels of testosterone induce vascular dysfunction via mROS generation and NLRP3 inflammasome activation. These events may contribute to increased cardiovascular risk.

Keywords: NLRP3 inflammasome; androgen receptor; reactive oxygen species; testosterone; vascular dysfunction.

Figure 1

Testosterone propionate treatment induces vascular dysfunction via NLRP3 inflammasome (in vivo experiments). Concentration-response curves to phenylephrine - PE (A) and acetylcholine - ACh (B) were performed in aortic rings; NLRP3 (C) and caspase-1 (D) expression was determined in thoracic aortas; IL-1β levels (E) in the serum of WT and NLRP3^−/− mice treated with testosterone propionate (10 mg/Kg for 30 days). Data are expressed as mean ± SEM (n = 3–10). *p < 0.05 vs. WT_Vehicle; ^#p < 0.05 vs. WT_TP.

Figure 2

Testosterone induces vascular dysfunction via NLRP3 inflammasome (in vitro experiments). Concentration-response curves to phenylephrine - PE (A) and acetylcholine - ACh (B); NLRP3 (C), caspase-1 (D) and IL-1β levels (E) expression, all determined in thoracic aortas incubated with testosterone (10⁻⁶ M for 2 h) from WT and NLRP3^−/− mice. Data are expressed as mean ± SEM (n = 3–10). *p < 0.05 vs. WT_Vehicle; ^#p < 0.05 vs. WT_Testo.

Figure 3

Pharmacological inhibition of NLRP3 inflammasome and androgen receptor prevents testosterone-induced vascular dysfunction. Concentration-response curves to phenylephrine - PE (A,C) and acetylcholine - ACh (B,D) were performed in aortic rings incubated with vehicle (WT_Vehicle) or testosterone (10⁻⁶ M for 2 h) (WT_Testo). The effects of MCC950 (10⁻⁶ M for 30 min) and Flutamide [Flu (10⁻⁵ M for 30 min)] on testosterone-induced vascular changes are shown in (A,B) - WT_MCC950 and WT_MCC950+Testo - and (C,D) - WT_Flu and WT_Flu+Testo - respectively. Data are expressed as mean ± SEM (n = 3–10). *p < 0.05 vs. WT_Vehicle; ^#p < 0.05 vs. WT_Testo.

Figure 4

Supraphysiological testosterone levels induce vascular generation of reactive oxygen species. Vascular ROS generation was measured by dihydroethidine in aortas from WT_Vehicle, WT_TP, NLRP3^−/−_Vehicle and NLRP3^−/−_TP mice (A); superoxide anion generation – measured by lucigenin in vessels incubated with Testo or vehicle (WT_Vehicle, WT_Testo, NLRP3^−/−_Vehicle and NLRP3^−/−_Testo) (B); effects of MCC950 (C), Flu (D) and CCCP (E) on testosterone effects in aortas isolated from WT mice and incubated with vehicle or testosterone. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs. WT_Vehicle; ^#p < 0.05 vs. WT_TP; ^#p < 0.05 vs. WT_Testo.

Figure 5

Testosterone induces vascular dysfunction via the generation of mitochondrial reactive oxygen species. Concentration-response curves to phenylephrine - PE (A) and acetylcholine - ACh (B) were performed in aortic rings incubated with testosterone (10⁻⁶ M for 2 h) or vehicle isolated from WT mice. The effects of CCCP (10⁻⁶ M for 30 min) on testosterone effects were determined. Data are expressed as mean ± SEM (n = 3–10). *p < 0.05 vs. WT_Vehicle; ^#p < 0.05 vs. WT_Testo.