Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G(i) (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G(q) (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1748-1716.2009.01964.x

Endothelial dysfunction and vascular disease

Polyphenols are found ubiquitously in plants and their regular consumption has been associated with a reduced risk of a number of chronic diseases, including cancer, cardiovascular disease (CVD) and neurodegenerative disorders. Rather than exerting direct antioxidant effects, the mechanisms by which polyphenols express these beneficial properties appear to involve their interaction with cellular signaling pathways and related machinery that mediate cell function under both normal and pathological conditions. We illustrate that their interactions with two such pathways, the MAP kinase (ERK, JNK, p38) and PI3 kinase/Akt signaling cascades, allow them to impact upon normal and abnormal cell function, thus influencing the cellular processes involved in the initiation and progression of cancer, CVD and neurodegeneration. For example, their ability to activate ERK in neurons leads to a promotion of neuronal survival and cognitive enhancements, both of which influence the progression of Alzheimer's disease, whilst ERK activation by polyphenols in vascular endothelial cells influences nitric oxide production, blood pressure and ultimately CVD risk. The main focus of this review is to provide an overview of the role that polyphenols play in the prevention of cancer, cardiovascular disease and neurodegeneration. We present epidemiological data, human intervention study findings, as well as animal and in vitro studies in support of these actions and in each case we consider how their actions at the cellular level may underpin their physiological effects.

Polyphenols and Human Health: Prevention of Disease and Mechanisms of Action

Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

Endothelial dysfunction and vascular disease – a 30th anniversary update

Background— Population-based studies suggest a reduced incidence of morbidity and mortality from coronary heart disease caused by moderate and regular consumption of red wine. Endothelial nitric oxide (NO) is a pivotal vasoprotective molecule. This study examines the influence of red wine polyphenols on the regulation of endothelial nitric oxide synthase (eNOS) expression and subsequent NO synthesis, focusing on the putative long-lasting antiatherosclerotic effects of red wine. Methods and Results— Treatment (20 hours) of human umbilical vein endothelial cells (HUVECs) and of the HUVEC-derived cell line EA.hy926 with a alcohol-free red wine polyphenol extract (RWPE) led to a concentration-dependent (100 to 600 μg/mL), significant increase in NO release (up to 3.0-fold/HUVEC and 2.0-fold/EA.hy926) as shown by use of the fluorescent probe DAF-2. This effect was corroborated by the [14C]l-arginine/l-citrulline conversion assay in intact EA.hy926 cells. RWPE (20 hours, 100 to 600 μg/mL) also significantly inc...

Red Wine Polyphenols Enhance Endothelial Nitric Oxide Synthase Expression and Subsequent Nitric Oxide Release From Endothelial Cells

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2801%2902901-5

Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein.

Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells invitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 µM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikertet al.,FEBS Lett 2001, 506:131–134) on aspects of validation procedures as well as limitations and pitfalls of this method.

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Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells in vitro.

Objective Accumulating data document protective effects of red wine in cardiovascular disease by improved nitric oxide bioavailability. Not all wines, however, seem to be equally effective. The aim of the present study was to elucidate the impact of grape cultivars, growing area and the vinification/fermentation process on the ability of red wine polyphenol extracts to increase human endothelial nitric oxide synthase (eNOS) promotor and enzyme activity. Methods We produced polyphenol extracts from a variety of red wines from all over the world and tested their influence on eNOS promotor activity by a luciferase reporter gene assay. Selected extracts were subjected to the L-arginine-L-citrulline conversion assay to confirm that enhanced eNOS transcription results in enhanced enzyme activity. Results We show that polyphenol extracts from wines of specific origin and grape cultivars vary strongly in their individual activity. However, when averaged, the activity could not be attributed to a specific grape cultivar or growing area. Resveratrol alone significantly enhanced eNOS promotor and enzyme activity, although at concentrations higher than those available in effective concentrations of red wine polyphenol extracts. Extracts of grape juice showed activity as well. Conclusions The biological activity of red wines on eNOS transcription depends neither on grape cultivars nor on growing area in general. Resveratrol may be the most promising ingredient in red wine polyphenol extracts, but does not account for the complete effect. A large part of the active components are genuine grape compounds, since extracts of grape juice that did not undergo a fermentation process also showed activity.

Activation of endothelial nitric oxide synthase by red wine polyphenols: impact of grape cultivars, growing area and the vinification process.

Cardiovascular diseases are known as the major causes of death or disability in western countries. Decreased bioavailability of endothelial derived nitric oxide (NO) is recognized as an important promoter in cardiovascular disease. In vivo studies suggest that phytoestrogens, especially isoflavones from soy, enhance endothelium-dependent vasoreactivity. We hypothesized that isoflavones may affect the expression of endothelial-type nitric oxide synthase (eNOS) and thereby NO formation in vitro. Human EA.hy926 endothelial cells were treated with the soybean isoflavones biochanin A and formononetin and with their metabolites genistein and daidzein. eNOS promoter activity was examined by a luciferase reporter gene assay (20 h). Active eNOS was detected by quantifying conversion of L-arginine to L-citrulline and by measuring NO released from endothelial cells using the fluorescent probe DAF-2 (20-96 h).eNOS promoter activity increased in response to isoflavone treatment (20 h). NO and L-citrulline production by EA.hy926 cells rose up to 1.7-fold of control levels after stimulation with genistein for 48-96 h. From these results, we conclude that the suggested positive effects of soy isoflavones on vascular reactivity may be indeed mediated via a long-term effect on the eNOS system.

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Thomas R Räthel

Papers

Red Wine Polyphenols Enhance Endothelial Nitric Oxide Synthase Expression and Subsequent Nitric Oxide Release From Endothelial Cells

Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein.

Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells in vitro.

Activation of endothelial nitric oxide synthase by red wine polyphenols: impact of grape cultivars, growing area and the vinification process.

The soy isoflavone genistein induces a late but sustained activation of the endothelial nitric oxide-synthase system in vitro