IL-6 induces mitochondrial ROS production and blunts NO bioavailability in human aortic endothelial cells

Abstract

Chronic inflammation is a major contributor to the development of endothelial dysfunction. Circulating concentrations of the proinflammatory cytokine interleukin-6 (IL-6) have been shown to predict cardiovascular disease risk and are associated with the development of vascular dysfunction. However, the mechanisms that underlie inflammation-induced endothelial dysfunction are not fully understood. Vascular endothelial dysfunction is characterized by blunted nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), with mitochondrial ROS suggested to play a primary role. Therefore, we tested the hypothesis that IL-6 induces mitochondrial ROS production and blunts NO bioavailability in endothelial cells. To study the effect of IL-6, we treated the human aortic endothelial cells (HAECs) with IL-6, MitoTEMPOL (MT; a mitochondria-targeted antioxidant), and/or a nitric oxide synthase (NOS) inhibitor (l-NAME) with and without ACh stimulation. Results are expressed as means ± SD (n = 4 replicates), one-way ANOVA, and Bonferroni's post hoc tests were performed. IL-6 treatment resulted in greater mitochondrial ROS (IL-6: 2.94 ± 0.93 a.u.) when compared with the untreated cells (Control: 1 ± 0; P = 0.0021) and also blunted NO bioavailability at baseline (Control: 1 ± 0; IL-6: 0.57 ± 0.08 a.u. P = 0.0008) and with acetylcholine stimulation (Control Ach: 1.27 ± 0.09; IL-6 Ach: 0.60 ± 0.13 a.u. P < 0.0001). Scavenging mitochondrial ROS with MT restored NO bioavailability in the IL-6-treated cells (IL-6: 0.57 ± 0.08; IL-6 MT: 1.16 ± 0.20 a.u. P < 0.0001). These findings indicate that IL-6 has a direct effect on mitochondrial ROS in human aortic endothelial cells, which leads to reduced nitric oxide bioavailability.NEW & NOTEWORTHY In this study, we provide evidence that IL-6 induces mitochondrial ROS production, which impairs nitric oxide bioavailability in human aortic endothelial cells. This finding adds an important perspective to the existing literature on the mechanism by which IL-6 contributes to endothelial dysfunction.

Keywords: MitoTEMPOL; endothelium; inflammation; vascular dysfunction.

Figure 1:

Effect of IL-6 on mitochondrial ROS. HAECs were treated with IL-6 for 16 hours with or without MT and stained with MitoSOX red for 30 minutes. a) Representative confocal images showing mitochondrial ROS fluorescence (red) and nucleus (blue). b) Bars represent mitochondrial ROS mean fluorescence intensity normalized to the control. Scale bar 50μm. Data are representative of four independent experiments done under similar conditions.

Figure 2:

Effect of IL-6 on endothelial nitric oxide bioavailability. HAECs were treated with L-NAME to ensure the specificity of the nitric oxide detection reagent. a) Representative confocal images showing NO fluorescence (red) and nucleus (blue) in cells treated with or without L-NAME. Bars represent NO mean fluorescence intensity normalized to the control. b) Representative confocal images and quantification of mean fluorescence intensity normalized to the control. Scale bar 50μm. Data are representative of four independent experiments done under similar conditions.

Figure 3:

Effect of Stat3 inhibitor WP1066 on mitochondrial ROS. HAECs were treated with the Stat3 inhibitor WP1066 and stained with MitoSOX red for 30 minutes. a) Representative confocal images showing mitochondrial ROS fluorescence (red) and nucleus (blue). b) Bars represent mitochondrial ROS mean fluorescence intensity normalized to the control. Scale bar 50μm. Data are representative of three independent experiments done under similar conditions.