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

Distinct radiation responses after <i>in vitro</i> mtDNA depletion are potentially related to oxidative stress

<div><p>Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ⁰). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ⁰ cells, while decreased for both tumor ρ⁰ lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ⁰ cells, while reduced in A549 and 143B ρ⁰ cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ⁰ cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ⁰, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.</p></div

ROS and GSH levels at basal levels and 24 hours after 4Gy of irradiation.

<p><b>A.</b> ROS levels expressed in mean intensity at baseline and 24 hours after irradiation, relative to each parental celline at baseline conditions. <b>B.</b> Ratio of GSH:GSSG levels at baseline and at 24 hours after irradiation for parental and ρ⁰ cell lines. Data represents the mean ± SEM from at least 3 independent biological repeats. mtDNA depleted cells are indicated by the dashed bars. * p<0.05. <b>C.</b> mRNA expression of KEAP1 and SOD2 24 hours after irradiation, normalized to each parental at baseline. Data represents the mean + SEM from at least 2 independent biological repeats. * p<0.05.</p

Validation of mtDNA depleted cell lines.

<p><b>A.</b> qPCR assessed mtDNA copy number levels expressed in percentage normalized to each parental (left panel) and the doubling time for all investigated cell lines (right panel). <b>B.</b> Basal respiration expressed as oxygen consumption rate (OCR) in function of time (minutes). <b>C.</b> Stacked plot of mitochondrial (left panel) or glycolysis (right panel) stress test after measuring OCR or extracellular acidification rate (ECAR) respectively. <b>D.</b> Relative ATP levels in A.U. corrected for cell number (left panel) and L-Lactic acid levels in g/l corrected for cell number (right panel). Data represent the mean ± SEM from at least 3 independent biological experiments. * p<0.05, *** p<0.001.</p