Ramaroson Andriantsitohaina

Resistance to endotoxic shock as a consequence of defective NF‐κB activation in poly (ADP‐ribose) polymerase‐1 deficient mice

Poly (ADP‐ribose) polymerase‐1 is a nuclear DNA‐binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP‐1‐deficient cells are defective in NF‐κB‐dependent transcription activation, but not in its nuclear translocation, in response to TNF‐α. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF‐κB in macrophages from PARP‐1+/+ but not from PARP‐1−/− mice. PARP‐1‐deficient mice were extremely resistant to LPS‐induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF‐κB‐dependent accumulation of TNF‐α in the serum and a down‐regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP‐1 and NF‐κB, with consequences for the transcriptional activation of NF‐κB and a systemic inflammatory process.

Nitric oxide production and endothelium‐dependent vasorelaxation induced by wine polyphenols in rat aorta

The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium‐derived nitric oxide (NO), was also investigated. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre‐contracted to the same extent with noradrenaline (0.3 and 0.1 μm, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium‐denuded rings compared to those with functional endothelium. High concentrations of catechin (in the range of 10−1 g l−1) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium. The NO synthase inhibitor, Nω‐nitro‐l‐arginine‐methyl‐ester (l‐NAME, 300 μm) completely abolished the endothelium‐dependent but not the endothelium‐independent relaxations produced by all of the polyphenolic compounds. In contrast to superoxide dismutase (SOD, 100 u ml−1), neither RWPC nor leucocyanidol affected the concentration‐response curve for the NO donor, SIN‐1 (3‐morpholino‐sydnonimine) which also produces superoxide anion (O2−). In aortic rings with endothelium, RWPC (10−2 g l−1) produced a 7 fold increase in the basal production of guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP) which was prevented by l‐NAME (300 μm). Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe2+‐diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by l‐NAME (300 μm). These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium‐dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O2−. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium‐dependent vasorelaxing activity.

Wine Polyphenols Decrease Blood Pressure, Improve NO Vasodilatation, and Induce Gene Expression

The effects of short-term oral administration of red wine polyphenolic compounds on hemodynamic parameters and on vascular reactivity were investigated in rats. Endothelial function and vascular smooth muscle contractility were studied in association with the induction of gene expression in the vascular wall. Rats were treated daily for 7 days by intragastric administration of either 5% glucose or red wine polyphenolic compounds (20 mg/kg). Administration of these compounds produced a progressive decrease in systolic blood pressure, which became significantly different on day 4. Aortas from rats treated with red wine polyphenolic compounds displayed increased endothelium-dependent relaxation to acetylcholine that was related to increased endothelial NO activity and involved a mechanism sensitive to superoxide anion scavengers. However, no increase in whole-body oxidative stress has been observed in rats treated with red wine polyphenolic compounds, as shown by plasma glutathione assay. Also, in the aorta, red wine polyphenolic compounds increased the expression of cyclooxygenase-2 and increased the release of endothelial thromboxane A2, which compensated for the extraendothelial NO–induced hyporeactivity in response to norepinephrine, resulting from enhanced inducible NO synthase expression. The present study provides evidence that short-term oral administration of red wine polyphenolic compounds produces a decrease in blood pressure in normotensive rats. This hemodynamic effect was associated with an enhanced endothelium-dependent relaxation and an induction of gene expression (of inducible NO synthase and cyclooxygenase-2) within the arterial wall, which together maintain unchanged agonist-induced contractility. These effects of red wine polyphenolic compounds may be a potential mechanism for preventing cardiovascular diseases.

Endothelial Dysfunction Caused by Circulating Microparticles from Patients with Metabolic Syndrome

Microparticles are membrane vesicles that are released during cell activation and apoptosis. Elevated levels of microparticles occur in many cardiovascular diseases; therefore, we characterized circulating microparticles from both metabolic syndrome (MS) patients and healthy patients. We evaluated microparticle effects on endothelial function; however, links between circulating microparticles and endothelial dysfunction have not yet been demonstrated. Circulating microparticles and their cellular origins were examined by flow cytometry of blood samples from patients and healthy subjects. Microparticles were used either to treat human endothelial cells in vitro or to assess endothelium function in mice after intravenous injection. MS patients had increased circulating levels of microparticles compared with healthy patients, including microparticles from platelet, endothelial, erythrocyte, and procoagulant origins. In vitro treatment of endothelial cells with microparticles from MS patients reduced both nitric oxide (NO) and superoxide anion production, resulting in protein tyrosine nitration. These effects were associated with enhanced phosphorylation of endothelial NO synthase at the site of inhibition. The reduction of O2(-) was linked to both reduced expression of p47 phox of NADPH oxidase and overexpression of extracellular superoxide dismutase. The decrease in NO production was triggered by nonplatelet-derived microparticles. In vivo injection of MS microparticles into mice impaired endothelium-dependent relaxation and decreased endothelial NO synthase expression. These data provide evidence that circulating microparticles from MS patients influence endothelial dysfunction.

A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses

Our method for producing tissue‐engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue‐engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re‐expression of VSMC‐specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration‐response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1,B2, and P2Y1, and pyrimidinoceptors). Sustained agonist‐induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine‐ and caffeine‐sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.—L Heureux, N., Stoclet, J.‐C., Auger, F. A., Lagaud, G. J.‐L., Germain, L., Andriantsitohaina, R. A human tissue‐engineered vascular media: a new model for pharmacological studies of contractile responses. FASEB J. 15, 515‐524 (2001)

Red wine polyphenols increase calcium in bovine aortic endothelial cells: a basis to elucidate signalling pathways leading to nitric oxide production

The present study investigates the mechanisms by which polyphenolic compounds from red wine elicit Ca2+ mobilization in bovine aortic endothelial cells (BAECs). Two polyphenol‐containing red wine extracts, red wine polyphenolic compounds (RWPC) and Provinols™, and delphinidin, an anthocyanin were used. RWPC stimulated a Ca2+‐dependent release of nitric oxide (NO) from BAECs accounting for the relaxation of endothelium‐denuded rat aortic rings as shown by cascade bioassay. RWPC, Provinols™ and delphinidin increased cytosolic free calcium ([Ca2+]i), by releasing Ca2+ from intracellular stores and by increasing Ca2+ entry. The RWPC‐induced increase in [Ca2+]i was decreased by exposure to ryanodine (30 μM), whereas Provinols™ and delphinidin‐induced increases in [Ca2+]i were decreased by bradykinin (0.1 μM) and thapsigargin (1 μM) pre‐treatment. RWPC, Provinols™ and delphinidin‐induced increases in [Ca2+]i were sensitive to inhibitors of phospholipase C (neomycin, 3 mM; U73122, 3 μM) and tyrosine kinase (herbimycin A, 1 μM). RWPC, Provinols™ and delphinidin induced herbimycin A (1 μM)‐sensitive tyrosine phosphorylation of several intracellular proteins. Provinols™ released Ca2+ via both a cholera (CTX) and pertussis toxins (PTX)‐sensitive pathway, whereas delphinidin released Ca2+ only via a PTX‐sensitive mechanism. Our data contribute in defining the mechanisms of endothelial NO production caused by wine polyphenols including the increase in [Ca2+]i and the activation of tyrosine kinases. Furthermore, RWPC, Provinols™ and delphinidin display differences in the process leading to [Ca2+]i increases in endothelial cells illustrating multiple cellular targets of natural dietary polyphenolic compounds.

Vascular bed heterogeneity in age-related endothelial dysfunction with respect to NO and eicosanoids

Endothelial dysfunction has been described with ageing but the mechanisms responsible have not been clearly elucidated and might be different from one vessel to the other. This study assesses the relative contribution of endothelial nitric oxide (NO) and cyclo‐oxygenase (COX) metabolites in relaxation to acetylcholine with ageing in the aorta and the small mesenteric artery of the rat. In the aorta and branch II or III of superior mesenteric artery (SMA), endothelium‐dependent relaxation to acetylcholine was not different between 12–14 (adult) and 32‐week‐old rats whereas it was reduced at 70–100 (old) weeks of age. Despite an increased endothelial NO‐synthase protein expression, the NO‐synthase inhibitor, NG‐nitro‐L‐arginine‐sensitive component of relaxation decreased with ageing. In old rats, exposure to the COX inhibitor, indomethacin, but not the selective COX‐2 inhibitor, NS‐398, potentiated response to acetylcholine. The thromboxane A2/prostaglandin H2 receptor antagonist, GR 32191B enhanced relaxation to acetylcholine in aorta but it had no effect in SMA. Furthermore, acetylcholine increased thromboxane B2 production (enzymeimmunoassay) in aorta but not in SMA. Finally, Western blot analysis showed enhanced expression of COX‐1 and 2 in the two arteries with ageing. These results suggest that the decrease in acetylcholine‐induced relaxation with ageing involves reduced NO‐mediated dilatation and increased generation of vasoconstrictor prostanoids most likely from COX‐1. They also point out vascular bed heterogeneity related to the nature of prostanoids involved between the aorta (i.e., thromboxane A2) and the SMA (unidentified) arteries even though increased expression of COX occurs in both vessels.

Red Wine Polyphenolic Compounds Inhibit Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells by Preventing the Activation of the p38 Mitogen-Activated Protein Kinase Pathway

Objective—Moderate consumption of red wine has a beneficial effect on the cardiovascular system. This study examines whether red wine polyphenolic compounds (RWPCs) affect vascular endothelial growth factor (VEGF) expression, a major angiogenic and proatherosclerotic factor in vascular smooth muscle cells (VSMCs). Methods and Results—VEGF mRNA expression was assessed by Northern blot analysis and the release of VEGF by immunoassay in cultured VSMCs. Short-term and long-term exposure of VSMCs to RWPCs inhibited VEGF mRNA expression and release of VEGF in response to platelet-derived growth factor AB (PDGFAB), transforming growth factor-&bgr;1, or thrombin. The PDGFAB-induced expression of VEGF was markedly reduced by SB203580 (inhibitor of p38 mitogen-activated protein kinase [MAPK]), antioxidants, and diphenylene iodonium (inhibitor of flavin-dependent enzymes), slightly reduced by PD98059 (inhibitor of MEK), and not significantly affected by wortmannin (inhibitor of PI-3-kinase) and L-JNKI (inhibitor of JNK). Short-term and long-term treatment of VSMCs with RWPCs markedly reduced PDGFAB-induced production of reactive oxygen species and phosphorylation of p38 MAPK. Conclusions—These data indicate that RWPCs strongly inhibit growth factor–induced VEGF expression in VSMCs by preventing the redox-sensitive activation of the p38 MAPK pathway. The potential antiangiogenic and antiatherosclerotic properties of RWPCs are likely to contribute to cardiovascular protection by preventing the development of atherosclerotic lesions.

Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis

Epidemiological studies have suggested that moderate consumption of natural dietary polyphenolic compounds might reduce the risk of cardiovascular disease and also protect against cancer. The present study investigates the effects of delphinidin, an anthocyanin present in red wine, on bovine aortic endothelial cells apoptosis. Based on flow cytometry, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling analysis and detection of mitochondrial cytochrome c release, we show that delphinidin (10−2 g l−1) alone had no effect either on necrosis or on apoptosis, but it significantly reduced apoptosis elicited by actinomycin D (1 μg ml−1, 24 h) and 7β‐hydroxycholesterol (10 μg ml−1, 18 h). The protective effect of delphinidin was abolished by inhibitors of nitric oxide‐synthase (NOS) (L‐NA, 100 μM and SMT, 100 μM), guanylyl cyclase (ODQ, 100 μM) and MAP kinase (PD98059, 30 μM). Western blot analysis and protein detection by confocal microscopy demonstrate that the antiapoptotic effect of delphinidin was associated with an increased endothelial NOS expression mediated by a MAP kinase pathway. Finally, delphinidin alone had no effect on cytosolic‐free calcium ([Ca2+]i), but normalized the changes in [Ca2+]i produced by actinomycin D towards the control values, suggesting that the antiapoptotic effect of delphinidin is associated with the maintenance of [Ca2+]i in the physiological range. All of the observed effects of delphinidin may preserve endothelium integrity, the alteration of which lead to pathologies including cardiovascular diseases, such as atherosclerosis, and is often associated with cancers. In conclusion, the protective effect of delphinidin against endothelial cell apoptosis contributes to understand the potential benefits of a consumption rich in polyphenols.

Delphinidin inhibits endothelial cell proliferation and cell cycle progression through a transient activation of ERK-1/-2

Epidemiological studies have shown that a diet rich in fruits and vegetables might reduce the risk of cardiovascular diseases and protect against cancer by mechanisms that have not been elucidated yet. This study was aimed to define the effect of delphinidin, a vasoactive polyphenol belonging to the class of anthocyanin, on bovine aortic endothelial cells (BAECs) proliferation. Delphinidin inhibited serum- and vascular endothelium growth factor-induced BAECs proliferation. This antiproliferative effect of delphinidin, is triggered by ERK-1/-2 activation, independent of nitric oxide pathway and is correlated with suppression of cell progression by blocking the cell cycle in G(0)/G(1) phase. Furthermore, suppression of cell cycle progression is associated with the modulation of the mitogenic signaling transduction cascade. This includes over-expression of caveolin-1 and p21(WAF1/Cip1) and down-expression of Ras and cyclin D1. In conclusion, the antiproliferative effect of delphinidin may be of importance in preventing both plaque development and stability in atherosclerosis and tumor dissemination in cancer.