Endothelial microparticles prevent lipid-induced endothelial damage via Akt/eNOS signaling and reduced oxidative stress

Abstract

Endothelial microparticles (EMPs) are endothelium-derived submicron vesicles that are released in response to diverse stimuli and are elevated in cardiovascular disease, which is correlated with risk factors. This study investigates the effect of EMPs on endothelial cell function and dysfunction in a model of free fatty acid (FFA) palmitate-induced oxidative stress. EMPs were generated from TNF-α-stimulated HUVECs and quantified by using flow cytometry. HUVECs were treated with and without palmitate in the presence or absence of EMPs. EMPs were found to carry functional eNOS and to protect against oxidative stress by positively regulating eNOS/Akt signaling, which restored NO production, increased superoxide dismutase and catalase, and suppressed NADPH oxidase and reactive oxygen species (ROS) production, with the involvement of NF-erythroid 2-related factor 2 and heme oxygenase-1. Conversely, under normal conditions, EMPs reduced NO release and increased ROS and redox-sensitive marker expression. In addition, functional assays using EMP-treated mouse aortic rings that were performed under homeostatic conditions demonstrated a decline in endothelium-dependent vasodilatation, but restored the functional response under lipid-induced oxidative stress. These data indicate that EMPs harbor functional eNOS and potentially play a role in the feedback loop of damage and repair during homeostasis, but are also effective in protecting against FFA-induced oxidative stress; thus, EMP function is reflected by the microenvironment.-Mahmoud, A. M., Wilkinson, F. L., McCarthy, E. M., Moreno-Martinez, D., Langford-Smith, A., Romero, M., Duarte, J., Alexander, M. Y. Endothelial microparticles prevent lipid-induced endothelial damage via Akt/eNOS signaling and reduced oxidative stress.

Keywords: Nrf2; endothelial dysfunction; extracellular microvesicles.

Figure 1.

EMPs express a functional eNOS, elevate NO in palmitate (Pal)-induced HUVECs, and increase phosphorylation levels of Akt and eNOS. A) EMPs were incubated with l-arginine for 5 min at 37°C, followed by the addition of DAF-2, and NO production was determined. EMPs produce eNOS-derived NO in a concentration-dependent manner, which is inhibited by the NOS inhibitor, L-NAME. The difference between fluorescence signal with and without L-NAME is considered NO production. B) peNOS^Ser1177 expression in EMPs and HUVECs as controls. C) HUVECs that were incubated with EMPs for 3 h showed no changes in NO production, whereas EMPs prevented the Pal-induced decline in NO production. D) Pal and EMPs diminish NO production. Treatment with either EMPs for 24 h with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal) or with EMPs and Pal for 24 h (24 h EMPs/Pal) protects against Pal-induced reduction in NO production. Results are means ± sem; n = 8–12. E, F) Treatment of HUVECs with 100 µM Pal for either 3 or 24 h decreases protein phosphorylation of Akt (E) and eNOS at Ser1177 (F). EMPs up-regulate Akt and eNOS phosphorylation in HUVECs that are treated with either EMPs for 24 h with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal) or with EMPs and Pal for 24 h (24 h EMPs/Pal). Results are means ± sem; n = 6 and analyzed using 1-way ANOVA. AUC, area under curve; ns, nonsignificant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Effects of EMPs and/or palmitate (Pal) on ROS production and oxidative stress in HUVECs. A) EMPs produce no effect on ROS production from HUVECs after a 3-h treatment period, but exhibit a significant protective effect after Pal-induced oxidative stress. B) EMPs significantly increase ROS production after treatment for 24 h, but with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal), or with EMPs and Pal for either 3 or 24 h (24 h EMPs/Pal), EMPs protect against Pal-induced ROS production. C–E) HUVECs that were treated with Pal for 3 and 24 h demonstrated a significant increase in MDA, a marker of lipid peroxidation (C), and concomitant decrease in the activity of SOD (D) and CAT (E). EMPs prevent Pal-induced lipid peroxidation and diminished the activity of antioxidant enzymes in HUVECs that were treated with either EMPs for 24 h with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal) or with EMPs and Pal for 24 h (24 h EMPs/Pal). Results are means ± sem; n = 6–10 and analyzed using 1-way ANOVA. AU, arbitrary unit. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Effects of EMPs and/or palmitate (Pal) on NADPH oxidase in HUVECs. NADPH oxidase activity measured by lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase subunits, NOX-4, NOX-1, p22^phox, and p47^phox, at the level of mRNA by RT-PCR. Palmitate and EMPs (10⁶) increase both NADPH oxidase activity (A) and expression of the enzyme subunits (B). EMPs reduce Pal-induced NADPH oxidase activation and expression in HUVECs that are treated with either EMPs for 24 h with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal) or with EMPs and Pal for 24 h (24 h EMPs/Pal). Results are means ± sem; n = 6–8 and analyzed using 1-way ANOVA. RLU, relative luminescence units. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.

Effects of EMPs and/or palmitate (Pal) on Nrf2/ARE pathway in HUVECs. A–F) Effect of EMPs and/or Pal treatment on HUVECs with regard to Nrf2 (A, D), NQO-1 (B, E), and HO-1 (C, F) levels was determined by using quantitative RT-PCR and Western blotting. Treatment with either EMPs or Pal produces a significant decrease in both mRNA (A–C) and protein (D–F) expression of Nrf2, NQO1, and HO-1. EMPs increase expression of Nrf2, NQO1, and HO-1 in HUVECs that are treated with either EMPs for 24 h with the addition of Pal during the last 3 h (24 h EMPs/3 h Pal) or with EMPs and Pal for 24 h (24 h EMPs/Pal). Results are means ± sem; n = 6 and analyzed using 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Effects of EMPs and/or palmitate (Pal) on endothelium-dependent vasodilatation and NADPH oxidase in mouse aortas. A, B) Treatment of aortic rings with either 10⁶ EMPs or Pal for 24 h diminished endothelium-dependent vasodilator responses to ACh. EMPs at both 10⁵ (A) and 10⁶ (B) doses improve the Pal-reduced endothelium-dependent vasodilatation. C, D) ROS are involved in endothelial dysfunction induced by EMPs (C) and Pal (D). MitoQ and apocynin improve the impaired aortic relaxation in response to ACh, induced by EMPs or Pal. E) NADPH oxidase activity increases in aortic rings that are treated with either EMPs or Pal for 24 h. In the presence of Pal, EMPs decrease NADPH oxidase activity. Results are means ± sem; n = 8–12. Data in panels A–D were analyzed using 2-way ANOVA and in panel E using 1-way ANOVA. CT, control; RLU, relative luminescence units. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6.

Schematic diagram demonstrating the molecular mechanisms by which EMPs inhibit FFA-induced endothelial dysfunction. O₂^⋅−, superoxide radical; ONOO^⋅, peroxynitrite; GPx, glutathione peroxidase; GST, glutathione-S-transferase.