De nombreuses études ont indiqué que la consommation régulière d’aliments riches en polyphénols comme le vin rouge, le thé, ou les fruits est associée à une réduction du risque de pathologies cardiovasculaires chez l’homme et les animaux. L’effet bénéfique des polyphénols sur le système cardiovasculaire est dû, au moins en partie, à leur action directe sur les vaisseaux sanguins en améliorant la fonction endothéliale. En effet, de nombreuses études indiquent que les polyphénols induisent des relaxations dépendantes de l’endothélium dans les artères isolées en stimulant la formation endothéliale de monoxyde d’azote (NO). La comparaison des relaxations induites par 13 jus et purées de fruits différents dans les artères coronaires de porc a permis de sélectionner l’Aronia melanocarpa en raison de sa grande activité et de sa forte teneur en polyphénols. L’Aronia melanocarpa est un puissant inducteur de relaxations dépendantes de l’endothélium en stimulant la formation endothéliale de NO. Cette formation accrue de NO implique l’activation rédox-sensible de la voie Src/PI3-kinase/Akt qui va phosphoryler la NO synthase sur son site activateur entraînant une formation rapide de NO. A plus long terme, l’Aronia melanocarpa stimule l’expression de la NO synthase via un mécanisme rédox-sensible impliquant les voies PI3-kinase/Akt, JNK, et p38 MAPK, et entraînant la phosphorylation inactivatrice des facteurs de transcription FoxO1 et Fox3a; cet effet prévient la régulation négative de l’expression de la NO synthase endothéliale. En conclusion,nos études révèlent le potentiel d’Aronia melanocarpa à améliorer la protection vasculaire par la stimulation soutenue de la formation de NO. / Many studies indicated that the regular consumption of drink or food rich in polyphenols like red wine, green tea, fruits, vegetables and chocolate is associated with a reduction of the risk of cardiovascular pathologies in human and animals. The beneficial effect of polyphenols, well known as antioxidants, on the cardiovascular system is due at least partly to their direct action on the blood-vessels by improving the endothelial function. Indeed, many studies indicate that the polyphenols induce the endothelium-dependent vasorelaxation in the isolated arteries by stimulating the formation of endothelial nitric oxide (NO). Comparing the endothelium-dependent relaxations induced by 13 different fruit juices and purees in the isolated porcine coronary arteries, Aronia melanocarpa was selected due to its high activity and the highest polyphenol content. Aronia melanocarpa is a potent inducer of endothelium-dependent relaxation in coronary artery by stimulating the formation of endothelial NO. This increased formation of NO involves the redox-sensitive activation of the Src/PI3-kinase/Akt pathway leading to the phosphorylation of eNOS at the activation site, Ser1177, during the rapid formation. Further for the long-term, Aronia melanocarpa stimulates the expression of eNOS via a redox-sensitive mechanism involving PI3-kinase/Akt, JNK, p38 MAPK pathways and the subsequent inactivation of transcription factors FoxO1 and FoxO3a by phosphorylation; this effect prevents their negative regulation of eNOS expression. In conclusion, our studies reveal the potential of Aronia melanocarpa to improve vascular protection by stimulating in a constant way the formation of endothelial NO.

The underlying inflammatory storm in renal or diabetic disease may induce expression of inducible nitric oxide synthase (iNOS). Similarly, expression of iNOS or nitric oxide (NO) production in vascular smooth muscle cells (VSMCs) in a calcifying environment, may promote vascular calcification (VC) (Zaragoza et al., 2006). However, emerging data suggests that NO generated by either endothelial nitric oxide synthase (eNOS) or iNOS may protect VSMCs from VC (Kanno et al., 2008). Thus, the role of NO and its associated enzymes in the development of VC is unclear. The aim of this study was to identify whether NO produced by iNOS regulates calcification in VSMCs, and to further understanding of potential mechanisms that may mediate the actions of NO/iNOS. A significant and sustained production of NO by iNOS, which peaked at day 3 and declined thereafter was found in rat aortic smooth muscle cells (RASMCs) that were preactivated with lipopolysaccharide (LPS; 100μg ml-1) and interferon gamma (IFN-γ;100U ml-1) in the presence of calcification buffer (CB) containing calcium chloride (CaCl2; 7mM) and β-glycerophosphate (β-GP; 7mM). This was associated with formation of hydroxyapatite crystals (HA) or calcification plaques, observed via alizarin red staining (ARS) and/or fourier transform infrared (FT-IR) analysis. However, when RASMCs were incubated with the iNOS inhibitor GW274150 at 10 μM, together with LPS + IFN-γ + CB, HA crystal formation was abolished. When RASMCs were pretreated with diethylenetriamine/nitric oxide adduct (NOC 18) at either 30 or 50 μM for an hour prior to addition of CB, to generate NO; calcium levels were elevated leading to form HA crystals. However, the elevation of calcium caused by the presence of NO generated via iNOS, did not result in phosphorylation of mitogen activated protein kinases (p38 MAPK), extracellular signal-regulated kinases (Erks), and protein kinase B. Furthermore, there was a reduction of Runx2 levels (pro-calcific factor) which could be another pro-calcific factor involved in this mechanism. These findings suggest that NO may indeed play a fundamental role in calcification, enhancing mineralisation of smooth muscle cells. Furthermore, the expression of iNOS/ NO appears to be enhanced under conditions that favour calcification and these together may contribute to enhanced calcification with potential detrimental consequences in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3

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Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the United States. It is also well recognized that the incidence of CVD is substantially increased in postmenopausal women due to the loss of estrogen. Experimental and clinical data support vascular protective effects of estrogen by various mechanisms. However, administration of estrogen is also associated with an increased incidence of heart disease which limits its therapeutic potential. Given the demonstrated risks of conventional estrogen therapy, a search for novel, cost-effective, alternative vasoactive agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity. Genistein, a major soy isoflavone, may be one of those alternative agents because of its selective affinity to estrogen receptor-beta and various beneficial effects on CVD. However, the mechanism of the cardioprotective effects of genistein is still unclear. The objectives of this study were (1) to investigate the effect of genistein on the expression of endothelial nitric oxide synthase (eNOS) both in vitro and in vivo; (2) to define the mechanism by which genistein regulates eNOS expression; and, (3) to examine whether genistein protects against tumor necrosis factor-alpha (TNF-α)-induced apoptosis in human aortic endothelial cells (HAECs). The results demonstrated that genistein, at physiologically achievable concentrations (1-10 μM) in individuals consuming soy products, enhanced the expression of eNOS protein and subsequently elevated nitric oxie (NO) synthesis in both HAECs and human umbilical vein endothelial cells, concomitant with the increased eNOS mRNA expression (2.6-fold of control) and eNOS promoter activity, suggesting that genistein activates eNOS transcription. Furthermore, dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt kinase, protein kinase C or inhibition of typrosine kinases, but possibly through activating the cAMP/protein kinase A/cAMP responsive elemant binding protein pathway. These data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increase in eNOS expression and NO synthesis, thereby improving vascular homeostasis. We also found that genistein (5-10 μM) significantly inhibited TNF-α-induced apoptosis in HAECs as determined by caspase-3 activation, apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of anti-apoptotic Bcl-2 protein and its promoter activity that was ablated by TNF-α. Moreover, this anti-apoptotic effect of genistein was not mediated by extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptor. However, inhibition of p38 mitogen-activated protein kinase (p38) by SB203580 completely abolished the cytoprotective effect of genistein, suggesting that genistein acted through the p38-dependent pathway. Accordingly, stimulation of HAECs with genistein resulted in rapid and dose-dependent activation of p38. Unlike TNF-α which specifically activated p38α, genistein selectively induced phosphorylation of p38β, suggesting that p38β, but not p38α, is essential for the cytoprotective effect of genistein. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Taken together, the resuls of the present study suggest that genistein can act directly on vascular ECs, improves endothelium homeostasis by promoting eNOS expression and endothelial-derived NO synthesis through activating the cAMP/PKA/CREB cascade, and protects against TNF-α-induced apoptosis via activation of p38 β. These data potentially provide a basic mechanism underlying the physiological effects of genistein in the vasculature. / Ph. D.

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.