NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration

Abstract

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H₂O₂), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10^-7 M) with or without a NOX4 inhibitor [GLX351322 (10^-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O₂^-) and H₂O₂ production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H₂O₂ production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

Keywords: NADPH oxidase; NOX4; endothelial dysfunction; oxidative stress; testosterone.

Graphical abstract

Figure 1.

Testosterone induces Noxs family expression. Endothelial cells (HMECs and HUVECs) were treated with Testo [Testosterone (10⁻⁷ M) for 4 and 24 h] and Noxs gene and protein expression was analyzed in HMECs (A and B, respectively) and HUVECs (C and D, respectively). Data are expressed as means ± SE (number of replicates = 3 or 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control. HMECs, primary human mesenteric vascular endothelial cells; HUVECs, human umbilical vein endothelial cells.

Figure 2.

Testosterone increases ROS generation in a Noxs-dependent manner. Endothelial cells (HMECs and HUVECs) were treated with Testo [Testosterone (10⁻⁷ M)] at different times (1 to 60 min) and ROS generation was evaluated in HMECs (A) and HUVECs (D). HMECs (B and C) and HUVECs (E–J) were also exposed to Testo for 10 min in the presence of vehicle, Melittin (10⁻⁷ M, NOX5 inhibitor), Nifedipinde [Nife (10⁻⁸ M), L-type calcium channel blocker], NOXA1ds (10⁻⁵ M, NOX1 inhibitor), NOX1 siRNA, GLX351322 (10⁻⁴ M, NOX4 inhibitor), or NOX4 siRNA. ROS generation was determined by lucigenin assay. Data are expressed as means ± SE (number of replicates = 3–6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. HMECs, primary human mesenteric vascular endothelial cells; HUVECs, human umbilical vein endothelial cells; ROS, reactive oxygen species.

Figure 3.

Testosterone augments H₂O₂ via NOX4. Endothelial cells (HUVECs) were treated with Testo [Testosterone (10⁻⁷ M)] at various times (10–60 min) and hydrogen peroxide (H₂O₂) production was evaluated (A). Cells were also exposed to Testo for 60 min in the presence of vehicle or GLX351322 (10⁻⁴ M, NOX4 inhibitor) (B) or NOX4 siRNA (C). H₂O₂ production was determined by Amplex red assay. Data are expressed as means ± SE (number of replicates = 4–6). *P < 0.05, ***P < 0.001, ****P < 0.0001. HUVECs, human umbilical vein endothelial cells.

Figure 4.

Endothelial cell dysfunction associated with testosterone is further impaired by NOX4 inhibition (in vivo experiments). Hydrogen peroxide (H₂O₂) production (A), concentration-response curves to acetylcholine – ACh (B–D) were performed in the presence of vehicle or GLX351322 (10⁻⁴ M, NOX4 inhibitor) or superoxide dismutase/catalase mimetic (EUK134, 10⁻⁵ M) in aortic rings of WT mice treated with Testosterone [Testo (10 mg/kg for 30 days)]. Data are expressed as means ± SE (number of animals = 4). *P < 0.05 vs. WT-Vehicle; #P < 0.05 vs. WT-Testo. WT, wild type.

Figure 5.

Increased NOX4 in testosterone-treated cells is a protective mechanism in endothelial cell dysfunction. Endothelial cells (HUVECs) were treated with Testo [Testosterone (10⁻⁷ M)] for 24 h in the presence of vehicle or GLX351322 (10⁻⁴ M, NOX4 inhibitor) and eNOS activation (A and B), total eNOS expression (C), and NO (nitric oxide) formation (D and E) were evaluated. NO formation was determined by 5,6-diaminofluorescein diacetate (DAF) assay. Bradykinin (10⁻⁵ M) was used as positive control. Data are expressed as means ± SE (number of replicates = 4–8). **P < 0.01, ****P < 0.0001, ns = not significant. HUVECs, human umbilical vein endothelial cells.

Figure 6.

Increased NOX4 in testosterone-treated cells is a protective mechanism in EC dysfunction by endothelial cell migration. Endothelial cells (HUVECs) were treated with Testo [Testosterone (10⁻⁷ M)] for 24 h in the presence of vehicle or GLX351322 (10⁻⁴ M, NOX4 inhibitor) and migration (A and B). Cell migration was determined by Scratch and Transwell migration assay. Data are expressed as means ± SE (number of replicates = 3–4). *P < 0.05 vs Testo, **P < 0.01 vs Testo, ***P < 0.001 vs Control, and ****P < 0.0001 vs Control HUVECs, human umbilical vein endothelial cells.