Antje R. Weseler

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 mans 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

The flavanol (-)-epicatechin and its metabolites protect against oxidative stress in primary endothelial cells via a direct antioxidant effect

Accumulating evidence suggests that foods rich in flavanols decrease the risk of developing cardiovascular diseases. Attenuation of oxidative stress was suggested to contribute to the cardiovascular benefit of flavanols. Up to now it was unclear whether flavanol metabolites can also protect cells from oxidative stress. The aim of the present study was to determine the potential contribution of several glucuronidated, methylated and sulfated metabolites of (-)-epicatechin (EC) and (+)-catechin (Cat) to the protection of human vascular endothelial cells (HUVECs) against oxidative stress. The relative potency of the tested compounds to scavenge superoxide anion radicals showed that a free catechol moiety in the molecule is important for the direct antioxidant activity. EC and Cat (0.5, 1, 10µM) were potent radical scavengers and provided protection against intracellular oxidative stress induced by hydrogen peroxide. Although the metabolites provided less intracellular protection compared to EC and Cat, the tested methylated and glucuronidated metabolites reduced oxidative stress significantly in HUVECs. Our results indicate that the metabolites have a relevant contribution in the intracellular protection of EC and Cat against oxidative stress. Also, the direct antioxidant activity plays an important role in this protection.

Pleiotropic-acting nutrients require integrative investigational approaches: the example of flavonoids.

Assessment of the health effects of dietary interventions in humans poses a particular challenge to nutritional and clinical scientists. In contrast to drugs possessing a well-defined molecular mechanism of action, food-derived components act in subtle and pleiotropic ways by nature. Moreover, dietary compounds are mainly not intended to cure a disease but to prevent or beneficially affect situations when the physiology gets slightly out of homeostasis. By the example of a recent clinical pilot study, this paper illustrates an endeavor to find new strategies for the detection of health effects of flavonoids in the human vasculature. Integration of a meticulously selected cluster of relevant biomarkers eventually enabled the beneficial vascular health effects of flavonoids to be revealed. A critical appraisal of this approach for the future is provided.

The cocoa flavanol (−)-epicatechin protects the cortisol response

Various health benefits of the cocoa flavanol (-)-epicatechin (EC) have been attributed to its antioxidant and anti-inflammatory potency. In the present study we investigated whether EC is able to prevent deterioration of the anti-inflammatory effect of the glucocorticoid (GC) cortisol in the presence of oxidative stress. It was found that cortisol reduces inflammation in differentiated monocytes. Oxidative stress extinguishes the anti-inflammatory effect of cortisol, leading to cortisol resistance. EC reduces intracellular oxidative stress as well as the development of cortisol resistance. This further deciphers the enigmatic mechanism of EC by which it exerts its anti-inflammatory and antioxidant action. The observed effect of the cocoa flavanol EC will especially be of relevance in pathophysiological conditions with increased oxidative stress and consequential GC resistance and provides a fundament for the rational use of dietary antioxidants.

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

Protection against Chemotaxis in the Anti-Inflammatory Effect of Bioactives from Tomato Ketchup

The consumption of tomato products has been associated with a decreased risk for chronic inflammatory diseases. In this study, the anti-inflammatory potential of tomato ketchup was evaluated by studying the effect of tomato ketchup extracts and bioactives from tomato ketchup on human monocytes and vascular endothelial cells (HUVEC). HUVEC were pre-treated for 1 h with either individual bioactives (7.5 µM lycopene, 1.4 µM α-tocopherol or 55 µM ascorbic acid) or a combination of these three compounds, or with the hydrophilic or lipophilic tomato ketchup extracts or with the two extracts combined. After the pretreatment, the cells were washed and challenged with TNF-α (10 ng/ml) for 6 h. The medium was used for the determination of the release of cytokines and the chemotaxis of monocytes. Inflammatory protein expression and production were assayed with real-time RT-PCR and ELISA. It was found that tomato ketchup extracts significantly reduced gene expression and release of the pro-inflammatory cytokines TNF-α and IL-8 in HUVEC after the inflammatory challenge, whereas the release of the anti-inflammatory cytokine IL-10 was increased. Chemotaxis was effectively impeded as demonstrated by a reduced monocyte migration. This effect correlated with the reduction of IL-8 production in the presence of the test compounds and extracts. The results consistently emphasize the contribution of lycopene to the anti-inflammatory effect of tomato ketchup. Other compounds in tomato ketchup such as α-tocopherol and ascorbic acid appeared to strengthen the anti-inflammatory effect of lycopene. The tomato ketchup extracts subtly interfered with several inflammatory phases that inhibit chemotaxis. Such a pleotropic mode of action exemplifies its potential mitigation of diseases characterized by prolonged low grade inflammation.

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.

Inhibition of acute pulmonary and systemic inflammation by 1,7-dimethylxanthine.

The nuclear enzyme poly(ADP-ribose) polymerse-1 (PARP-1) has previously been reported to play an important role in lipopolysaccharide (LPS)-induced pulmonary inflammation and is highly activated in COPD patients. In the present study, the anti-inflammatory efficacy of a previously identified poly(ADP-ribose) polymerase-1 (PARP-1) inhibiting caffeine metabolite, 1,7-dimethylxanthine, was both in vivo as well as ex vivo evaluated. Orally administered 1,7-dimethylxanthine significantly attenuated lung myeloperoxidase-levels, transcription of IL-6, TNF-alpha, MIP1alpha and MIP2 genes as well as PAR-polymer formation in a mouse model of intratracheally LPS-induced acute pulmonary inflammation. Serum amyloid P component and plasma IL-6 were also lowered in 1,7-dimethylxanthine treated mice, indicating a reduced systemic inflammatory response. In addition, at 24h after LPS administration anti-inflammatory effects of 1,7-dimethylxanthine appeared more pronounced than those of the orally administered PARP-1 inhibitor 3-aminobenzamide. In the second model, in blood of COPD-patients and healthy controls ex vivo pre-incubated with a physiological concentration of 1,7-dimethylxanthine (10microM), LPS-induced production of the cytokines IL-6 and TNF-alpha was significantly suppressed. 1,7-Dimethylxanthine exerts anti-inflammatory effects, both in vivo mouse as well as ex vivo human. These results suggest that the PARP-1 inhibiting caffeine metabolite 1,7-dimethylxanthine may have therapeutic potential in pulmonary inflammatory diseases such as COPD.

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. Graphical abstract Figure. No Caption available. HighlightsCigarette smoke extract (CSE) enhances exosome release by airway epithelial cells.This is mediated by thiol‐reactive CSE components such as acrolein.CSE depletes cell surface thiols, which is sufficient to trigger exosome release.CSE‐induced exosome release can be prevented by N‐acetyl‐l‐cysteine and glutathione.

Food-Derived Bioactives Can Protect the Anti-Inflammatory Activity of Cortisol with Antioxidant-Dependent and -Independent Mechanisms

In chronic inflammatory diseases the anti-inflammatory effect of glucocorticoids (GCs) is often decreased, leading to GC resistance. Inflammation is related with increased levels of reactive oxygen species (ROS), leading to oxidative stress which is thought to contribute to the development of GC resistance. Plant-derived compounds such as flavonoids are known for their ability to protect against ROS. In this exploratory study we screened a broad range of food-derived bioactives for their antioxidant and anti-inflammatory effects in order to investigate whether their antioxidant effects are associated with the ability to preserve the anti-inflammatory effects of cortisol. The anti-inflammatory potency of the tested compounds was assessed by measuring the oxidative stress–induced GC resistance in human macrophage-like cells. Cells were pre-treated with H2O2 (800 µM) with and without bioactives and then exposed to lipopolysaccharides (LPS) (10 ng/mL) and cortisol (100 nM). The level of inflammation was deducted from the concentration of interleukin-8 (IL-8) in the medium. Intracellular oxidative stress was measured using the fluorescent probe 2′,7′-dichlorofluorescein (DCFH). We found that most of the dietary bioactives display antioxidant and anti-inflammatory action through the protection of the cortisol response. All compounds, except for quercetin, revealing antioxidant activity also protect the cortisol response. This indicates that the antioxidant activity of compounds plays an important role in the protection of the GC response. However, next to the antioxidant activity of the bioactives, other mechanisms also seem to be involved in this protective, anti-inflammatory effect.