Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan 11:2022:4413191.
doi: 10.1155/2022/4413191. eCollection 2022.

(-)-Epicatechin Alters Reactive Oxygen and Nitrogen Species Production Independent of Mitochondrial Respiration in Human Vascular Endothelial Cells

Daniel G Sadler  1 , Jonathan Barlow  2 , Richard Draijer  3 , Helen Jones  1 , Dick H J Thijssen  1 , Claire E Stewart  1
Affiliations

(-)-Epicatechin Alters Reactive Oxygen and Nitrogen Species Production Independent of Mitochondrial Respiration in Human Vascular Endothelial Cells

Daniel G Sadler et al. Oxid Med Cell Longev. .

Abstract

Introduction: Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction, and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPI's effects remain unclear. Objective(s). Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function.

Methods: HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting.

Results: Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL (P < 0.001). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P < 0.001). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL (P = 0.010). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL (P = 0.015 and P = 0.001, respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P = 0.035 and P = 0.011). Conclusion(s). EPI dose-dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

PubMed Disclaimer

Conflict of interest statement

Daniel G. Sadler, Jonathan Barlow, Helen Jones, Dick H. J. Thijssen, and Claire E. Stewart had no conflict of interest associated with this manuscript. Richard Draijer is employed by Unilever.

Figures

Figure 1
Figure 1
EPI does not cause vascular endothelial cell toxicity. HUVECs were treated with 0-20 μM EPI for 24 h. Data are means ± SEM, representative of 3 independent repeats with 3 replicates of each condition. Statistical significance was tested for by one-way ANOVA and Dunnett's test for multiple comparisons. αSignificant main effect of dose (P < 0.05).
Figure 2
Figure 2
EPI dose-dependently impacts RONS production in vascular endothelial cells. (a) MitoSOX oxidation rates in HUVECs after 24 h EPI treatment and 30 minutes incubation with or without antimycin A. (b) Mean unnormalized MitoSOX oxidation rates measured in 30-second intervals over 30 minutes. (c) CellROX oxidation in HUVECs after 24 h EPI treatment. (d) DAF-FM oxidation in HUVECs after 24 h EPI treatment measured by flow cytometry. Data are means ± SEM of three independent repeats with two replicates per treatment. Statistical significance was tested for by a two-way ANOVA, with dose and antimycin A as factors: αSignificant main effect of dose; δSignificant main effect of AA (P < 0.05). ∗P < 0.05 and ∗∗∗∗P < 0.0001.
Figure 3
Figure 3
Gene expression responses following acute EPI treatment. HUVECs were treated with 0, 5, and 10 μM EPI over 48 h and lysed for analysis of gene expression. (a) DRP1, (b) MFN2, (c) NRF2, and (d) SOD2. Data are means ± SEM from 3 independent experiments. Statistical significance was determined by a two-way ANOVA, with dose and time as factors. Multiple comparisons were performed by Dunnett's test to determine differences in gene expression between conditions.αMain effect of dose; βmain effect of time (P < 0.05); ∗P < 0.05, ∗∗P < 0.01.
Figure 4
Figure 4
EPI does not directly affect mitochondrial bioenergetics. (a) Mitochondrial bioenergetics of HUVECs following 24 h EPI treatment (0, 5, and 10 μM). (b) Coupling efficiency of oxidative phosphorylation. (c) Relative contribution of glycolytic (grey) and oxidative (black) ATP production to total ATP production rates. (d) Representative trace of oxygen consumption rates during the mitochondrial stress test. Data from 3 independent experiments are normalised to cell number (1 × 103) and presented as mean ± SEM. Max: maximal respiratory capacity; PL: proton leak; ADPphos: ADP phosphorylation; SRC: spare respiratory capacity.
Figure 5
Figure 5
EPI rapidly and transiently activates ERK1/2 signalling whilst blunting AMPK phosphorylation. (a) AMPKα phosphorylation at Thr172 in HUVECs in the absence (-; clear bars) or presence (+; green bars) of EPI. (b) ERK1/2 phosphorylation at Thr202/Tyr204. (c) eNOS phosphorylation at Ser1177. (d) Representative images of n = 3 independent experiments. Cell lysates were analysed by SDS-PAGE and western blotting with indicated antibodies. Data are expressed as means ± SEM; ∗P < 0.05 and ∗∗∗P < 0.001. αSignificant main effect of treatment; βsignificant main effect of time (P < 0.05).
Figure 6
Figure 6
Schematic of the potential mechanisms by which EPI exerts its biological effects in vascular endothelial cells. Solid arrows/lines signify EPI's mode of action demonstrated in this study. Dashed arrows represent the potential activity of EPI not examined in this study but reported previously.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. WHO. Cardiovascular Diseases. Accessed July 2021, https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases...
    1. Heitzer T., Schlinzig T., Krohn K., Meinertz T., Münzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation . 2001;104(22):2673–2678. doi: 10.1161/hc4601.099485. - DOI - PubMed
    1. Suwaidi J. A., Hamasaki S., Higano S. T., Nishimura R. A., Holmes D. R., Lerman A. Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. Circulation . 2000;101(9):948–954. doi: 10.1161/01.CIR.101.9.948. - DOI - PubMed
    1. Ungvari Z., Labinskyy N., Gupte S., Chander P. N., Edwards J. G., Csiszar A. Dysregulation of mitochondrial biogenesis in vascular endothelial and smooth muscle cells of aged rats. American Journal of Physiology-Heart and Circulatory Physiology . 2008;294(5):H2121–H2128. doi: 10.1152/ajpheart.00012.2008. - DOI - PubMed
    1. Gliemann L., Rytter N., Piil P., et al. The endothelial mechanotransduction protein platelet endothelial cell adhesion molecule-1 is influenced by aging and exercise training in human skeletal muscle. Frontiers in Physiology . 2018;9:p. 1807. doi: 10.3389/fphys.2018.01807. - DOI - PMC - PubMed

LinkOut - more resources

-