Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants

Introduction: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-L-cysteine (NAC) or glutathione (GSH). Methods: BEAS-2B cells were exposed for 24 h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63(+)CD81(+) bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. Results: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. Summary/conclusion: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein-and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment

Oxidative Stress and Vascular Function: Implications for Pharmacologic Treatments

Production of considerable amounts of reactive oxygen species (ROS) eventually leads to oxidative stress. A key role of oxidative stress is evident in the pathologic mechanisms of endothelial dysfunction and associated cardiovascular diseases. Vascular enzymes such as NADPH oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase are involved in the production of ROS. The question remains whether pharmacologic approaches can effectively combat the excessive ROS production in the vasculature. Interestingly, existing registered cardiovascular drugs can directly or indirectly act as antioxidants, thereby preventing the damaging effects of ROS. Moreover, new compounds targeting NADPH oxidases have been developed. Finally, food-derived compounds appear to be effective inhibitors of oxidative stress and preserve vascular function

Pleiotropic Benefit of Monomeric and Oligomeric Flavanols on Vascular Health - A Randomized Controlled Clinical Pilot Study

BACKGROUND: Cardiovascular diseases are expanding to a major social-economic burden in the Western World and undermine man's deep desire for healthy ageing. Epidemiological studies suggest that flavanol-rich foods (e.g. grapes, wine, chocolate) sustain cardiovascular health. For an evidenced-based application, however, sound clinical data on their efficacy are strongly demanded. METHODS: In a double-blind, randomized, placebo-controlled intervention study we supplemented 28 male smokers with 200 mg per day of monomeric and oligomeric flavanols (MOF) from grape seeds. At baseline, after 4 and 8 weeks we measured macro- and microvascular function and a cluster of systemic biomarkers for major pathological processes occurring in the vasculature: disturbances in lipid metabolism and cellular redox balance, and activation of inflammatory cells and platelets. RESULTS: In the MOF group serum total cholesterol and LDL decreased significantly (P ≤ 0.05) by 5% (n = 11) and 7% (n = 9), respectively in volunteers with elevated baseline levels. Additionally, after 8 weeks the ratio of glutathione to glutathione disulphide in erythrocytes rose from baseline by 22% (n = 15, P<0.05) in MOF supplemented subjects. We also observed that MOF supplementation exerts anti-inflammatory effects in blood towards ex vivo added bacterial endotoxin and significantly reduces expression of inflammatory genes in leukocytes. Conversely, alterations in macro- and microvascular function, platelet aggregation, plasma levels of nitric oxide surrogates, endothelin-1, C-reactive protein, fibrinogen, prostaglandin F2alpha, plasma antioxidant capacity and gene expression levels of antioxidant defense enzymes did not reach statistical significance after 8 weeks MOF supplementation. However, integrating all measured effects into a global, so-called vascular health index revealed a significant improvement of overall vascular health by MOF compared to placebo (P ≤ 0.05). CONCLUSION: Our integrative multi-biomarker approach unveiled the pleiotropic vascular health benefit of an 8 weeks supplementation with 200 mg/d MOF in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT00742287

Monomeric and oligomeric flavanols maintain the endogenous glucocorticoid response in human macrophages in pro-oxidant conditions in vitro

Chronic inflammation and oxidative stress are (sub)cellular processes that enhance each other and contribute to the genesis of many systemic pathologies. The endogenous glucocorticoid cortisol plays an important role in the physiological termination of a pro-inflammatory immune response. However, in conditions of pronounced oxidative stress the anti-inflammatory action of cortisol is impaired. Since grape seed-derived monomeric and oligomeric flavan-3-ols (MOF) have been shown to attenuate both inflammation and oxidative stress in vitro and in humans, we hypothesized that these compounds are able to maintain the anti-inflammatory activity of cortisol in immune cells in a pro-oxidant environment. In a glucocorticoid resistance model using human monocytes (THP-1 cell line) differentiated into macrophage-like cells we observed that exposure to 1 mM tertiary butyl hydroperoxide (t-BuOOH) for 4 h significantly hampered the anti-inflammatory action of cortisol assessed as attenuation of the interleukin (IL)-8 production. Under these conditions, the effects of MOF were assessed on pro-inflammatory cytokines expression, cortisol's anti-inflammatory action and on the expression of 115-hydroxysteroid dehydrogenase (1113-HSD) 1, which catalyzes intracellular conversion of cortisone to cortisol. MOF attenuated the gene expression of pro-inflammatory cytokines and prevented the decline of the antiinflammatory effect of cortisol in the presence of t-BuOOH. MOF also maintained the activity of histone deacetylase in the cell nucleus which is essential for cortisol's molecular action to terminate the transcription of proinflammatory genes. Moreover, MOF prevented the down-regulation of 11 beta-HSD1 gene expression in this prooxidant cellular environment. Taken together our data suggest that MOF contribute to maintain the anti-inflammatory action of cortisol under pro-oxidant conditions via preservation of the intracellular availability of bioactive cortisol and cortisol-mediated termination of pro-inflammatory gene transcription. These findings provide novel insights in how MOF may enhance the ability to adapt, which is of particular relevance for their rational use as dietary supplement to maintain health

Flavanols exert cardiovascular health benefits in humans by altering gene transcription levels in white blood cells

International audienc

Time in Redox Adaptation Processes: From Evolution to Hormesis

Life on Earth has to adapt to the ever changing environment. For example, due to introduction of oxygen in the atmosphere, an antioxidant network evolved to cope with the exposure to oxygen. The adaptive mechanisms of the antioxidant network, specifically the glutathione (GSH) system, are reviewed with a special focus on the time. The quickest adaptive response to oxidative stress is direct enzyme modification, increasing the GSH levels or activating the GSH-dependent protective enzymes. After several hours, a hormetic response is seen at the transcriptional level by up-regulating Nrf2-mediated expression of enzymes involved in GSH synthesis. In the long run, adaptations occur at the epigenetic and genomic level; for example, the ability to synthesize GSH by phototrophic bacteria. Apparently, in an adaptive hormetic response not only the dose or the compound, but also time, should be considered. This is essential for targeted interventions aimed to prevent diseases by successfully coping with changes in the environment e.g., oxidative stress

The effect of tomato ketchup extracts and lycopene (7.5 µM), α-tocopherol (1.4 µM) and ascorbic acid (55 µM) on gene expression of E-selectin and ICAM-1 in HUVEC after an inflammatory challenge.

<p>Data are presented as mean ± S.E.M. of 3 triplicates,* significantly lower (<i>P</i>≤0.05) than the control (only the inflammatory challenge). Mix AOX: the combination of lycopene, α-tocopherol and ascorbic acid; Hydro: hydrophilic tomato ketchup extract; Lipo: lipophilic tomato ketchup extract; Mix extracts: the combination of the hydrophilic and lipophilic tomato ketchup extracts.</p

The effect of tomato ketchup extracts and lycopene (7.5 µM), α-tocopherol (1.4 µM) and ascorbic acid (55 µM) on gene expression of TNF-α (panel A) IL-8 (panel B) and IL-10 (panel C) and the concentration of these cytokines in the medium of the HUVEC after an inflammatory challenge.

<p>For TNF-α, the effect on gene expression showed a significant correlation with the effect on protein production (panel A). A comparable correlation was also seen for IL-8 (panel B), but not for IL-10 (panel C). Data are presented as mean ± S.E.M. of 3 triplicates. The effect of the pretreatments was significantly different (<i>P</i>≤0.05) from the control (only the inflammatory challenge), except for the hydrophilic extract on the gene expression of TNF-α, the effect of the hydrophilic extract and ascorbic acid on the IL-10 concentration, and the effect of all the pretreatments on the gene expression of IL10. Mix AOX: the combination of lycopene, α-tocopherol and ascorbic acid; Hydro: hydrophilic tomato ketchup extract; Lipo: lipophilic tomato ketchup extract; Mix extracts: the combination of the hydrophilic and lipophilic tomato ketchup extracts.</p