Chiara Stranieri

Nebivolol decreases oxidative stress in essential hypertensive patients and increases nitric oxide by reducing its oxidative inactivation.

Objective To obtain further insight into the mechanism underlying the vasodilator effect of nebivolol. Since oxidative inactivation of nitric oxide (NO) is regarded as an important cause of its decreased biological activity, we studied (1) the effect of nebivolol on some oxidative parameters in essential hypertensive patients; (2) the effect of plasma of nebivolol-treated patients on reactive oxygen species production and NO availability in endothelial cells. Methods A total of 20 healthy subjects and 20 matched essential hypertensive patients treated with atenolol or nebivolol according to a double-blind, randomized design participated in the study. We measured low-density lipoprotein (LDL) and plasma hydroperoxides, 8-isoprostanes, oxidized LDL, susceptibility of LDL to oxidation (lag phase) and LDL vitamin E and the effect of plasma of nebivolol- and atenolol-treated patients on reactive oxygen species production and NO availability in endothelial cells exposed to oxidative stress. Results In hypertensive patients, nebivolol and atenolol significantly reduced blood pressure values after 4 weeks of treatment. Plasma and LDL hydroperoxides, plasma 8-isoprostanes, plasma ox-LDL and LDL lag phase were significantly improved only in the patients receiving nebivolol compared with the atenolol group. Similarly there was a reduction of reactive oxygen species (ROS) and O2•− concentration in endothelial cells exposed to oxidative stress after incubation of the cells with plasma of the patients enrolled in the trial only in the patients receiving nebivolol compared to atenolol group. Furthermore, the reduction of basal and stimulated NO induced by oxidative stress in endothelial cells was significantly lower in the patients receiving nebivolol compared to atenolol group. Conclusions The findings of the present study indicate that nebivolol, through its antioxidant properties, increases NO also by decreasing its oxidative inactivation.

PlA polymorphism and platelet reactivity following clopidogrel loading dose in patients undergoing coronary stent implantation.

The PlA polymorphism (Leu33Pro) of the platelet glycoprotein (GP) IIIa gene has been suggested to play an important role in coronary thrombosis. In vitro studies have shown differences for this polymorphism in platelet sensitivity towards antiplatelet drugs (aspirin and abciximab), suggesting a pharmacogenetic modulation. The aim of the study was to assess the modulatory effect of the PlA polymorphism on clopidogrel-induced antiplatelet effects in 38 patients undergoing coronary stent implantation receiving a 300 mg clopidogrel loading-dose. Platelet reactivity was assessed as GPIIb/IIIa activation and P-selectin expression in platelets stimulated with 2 μmol/l adenosine diphosphate using whole blood flow cytometry. The distribution of the homozygous PlA1/A1 and heterozygous PlA1/A2 genotypes were 74 and 26%, respectively. PlA2 carriers had a higher degree of GPIIb/IIIa activation (P = 0.05) and P-selectin expression (P = 0.02) during the overall study time course and a lower antiplatelet effect to a 300 mg clopidogrel loading-dose up to 24 h following intervention (P < 0.05). In conclusion, the PlA polymorphism of the GPIIIa gene modulates platelet reactivity towards clopidogrel front loading in patients undergoing coronary stenting. This suggests the need for individualized antithrombotic regimens to optimally inhibit platelet reactivity.

Nebivolol treatment reduces serum levels of asymmetric dimethylarginine and improves endothelial dysfunction in essential hypertensive patients.

BACKGROUND This study was conducted to evaluate (i) the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on plasma concentration of asymmetric dimethylarginine (ADMA) and on flow-mediated dilation (FMD) in essential hypertensive patients; (ii) the effect of serum derived from the treated hypertensive patients on ADMA and on dimethylarginine dimethylaminohydrolase 2 (DDAH2), the enzyme that selectively degrades ADMA, in human umbilical vein endothelial cells (HUVECs). METHODS Forty healthy subjects and 40 matched essential hypertensive patients treated with atenolol and nebivolol according to a double-blind, randomized design participated in the study. Evaluation of brachial artery (BA) reactivity was performed by a longitudinal B-mode scan of the right BA. ADMA and L-arginine were measured by high-performance liquid chromatography. DDAH2 expression and endothelial nitric oxide synthase activity (eNOS) were also evaluated in HUVECs. RESULTS ADMA levels were significantly decreased and FMD increased only in patients receiving nebivolol (P < 0.01). Furthermore, in nebivolol group, we found a significant correlation between changes in ADMA levels and changes in FMD (P < 0.01). Sera derived from patients treated with nebivolol but not with atenolol decreased ADMA and increased DDAH2 expression and eNOS activity (P < 0.001) in HUVECs. CONCLUSIONS The results of this study demonstrate that the improvement of endothelial dysfunction induced by nebivolol in hypertensive patients may be related to its effect on circulating ADMA levels. Although the mechanism by which nebivolol reduces circulating ADMA in hypertensive patients remains unclear, our ex vivo results suggest that the upregulation of DDAH2 expression may have a role.

Cigarette Smoking Blocks the Protective Expression of Nrf2/ARE Pathway in Peripheral Mononuclear Cells of Young Heavy Smokers Favouring Inflammation

Cigarette smoking is an important risk factor for atherosclerosis, a chronic inflammatory disease. However the underlying factors of this effect are unclear. It has been hypothesized that water-soluble components of cigarette smoke can directly promote oxidative stress in vasculature and blood cells. Aim of this study was to study the relationship between oxidative stress and inflammation in a group of young smokers. To do this we evaluated: 1) the oxidation products of phospholipids (oxPAPC) in peripheral blood mononuclear cells (PBMC); 2) their role in causing PBMC reactive oxygen species (ROS) generation and changes in GSH; 3) the expression of the transcription factor NF-E2-related factor 2 (Nrf2) and of related antioxidant genes (ARE); 4) the activation of NF-kB and C-reactive protein (CRP) values. We studied 90 healthy volunteers: 32 non-smokers, 32 moderate smokers (5–10 cigarettes/day) and 26 heavy smokers (25–40 cigarettes/day). OxPAPC and p47phox expression, that reasonably reflects NADPH oxidase activity, were higher in moderate smokers and heavy smokers than in non-smokers (p<0.01), the highest values being in heavy smokers (p<0.01). In in vitro studies oxPAPC increased ROS generation via NADPH oxidase activation. GSH in PBMC and plasma was lower in moderate smokers and heavy smokers than in non-smokers (p<0.01), the lowest values being in heavy smokers (p<0.01). Nrf2 expression in PBMC was higher in moderate smokers than in non-smokers (p<0.01), but not in heavy smokers, who had the highest levels of NF-kB and CRP (p<0.01). In in vitro studies oxPAPC dose-dependently increased NF-kB activation, whereas at the highest concentrations Nrf2 expression was repressed. The small interference (si) RNA-mediated knockdown of NF-κB/p65 increased about three times the expression of Nrf2 stimulated with oxPAPC. Cigarette smoke promotes oxPAPC formation and oxidative stress in PBMC. This may cause the activation of NF-kB that in turn may participate in the negative regulation of Nrf2/ARE pathway favouring inflammation.

Endoplasmic reticulum stress and Nrf2 signaling in cardiovascular diseases

Various cellular perturbations implicated in the pathophysiology of human diseases, including cardiovascular and neurodegenerative diseases, diabetes mellitus, obesity, and liver diseases, can alter endoplasmic reticulum (ER) function and lead to the abnormal accumulation of misfolded proteins. This situation configures the so-called ER stress, a form of intracellular stress that occurs whenever the protein-folding capacity of the ER is overwhelmed. Reduction in blood flow as a result of atherosclerotic coronary artery disease causes tissue hypoxia, a condition that induces protein misfolding and ER stress. In addition, ER stress has an important role in cardiac hypertrophy mainly in the transition to heart failure (HF). ER transmembrane sensors detect the accumulation of unfolded proteins and activate transcriptional and translational pathways that deal with unfolded and misfolded proteins, known as the unfolded protein response (UPR). Once the UPR fails to control the level of unfolded and misfolded proteins in the ER, ER-initiated apoptotic signaling is induced. Furthermore, there is considerable evidence that implicates the presence of oxidative stress and subsequent related cellular damage as an initial cause of injury to the myocardium after ischemia/reperfusion (I/R) and in cardiac hypertrophy secondary to pressure overload. Oxidative stress is counterbalanced by complex antioxidant defense systems regulated by a series of multiple pathways, including the UPR, to ensure that the response to oxidants is adequate. Nuclear factor-E2-related factor (Nrf2) is an emerging regulator of cellular resistance to oxidants; Nrf2 is strictly interrelated with the UPR sensor called pancreatic endoplasmic reticulum kinase. A series of studies has shown that interventions against ER stress and Nrf2 activation reduce myocardial infarct size and cardiac hypertrophy in the transition to HF in animals exposed to I/R injury and pressure overload, respectively. Finally, recent data showed that Nrf2/antioxidant-response element pathway activation may be of importance also in ischemic preconditioning, a phenomenon in which the heart is subjected to one or more episodes of nonlethal myocardial I/R before the sustained coronary artery occlusion.

A1298C methylenetetrahydrofolate reductase mutation and coronary artery disease: relationships with C677T polymorphism and homocysteine/folate metabolism.

Abstract 5, 10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in homocysteine/methionine metabolism. The most-studied C677T polymorphism in the MTHFR gene results in a thermolabile variant with reduced activity, and is associated with increased levels of total plasma homocysteine, a risk factor for coronary artery disease. A new mutation in the MTHFR gene (A1298C) has also been reported to lower enzyme activity. Whether A1298C is a risk factor for coronary artery disease, separately or in combination with C677T, and/or relative to total plasma homocysteine and folate status, is unclear to date. We evaluated this hypothesis in 470 angiographically characterized subjects, 302 with coronary artery disease, and 168 with normal coronary arteries. The frequency of the 1298C allele was 0.33 and that of combined heterozygosity 0.315. No difference was found in the frequency of the genotypes or when analyzed for combined heterozygosity between patients with coronary artery disease and normals. Independent of folate status, the 1298C allele was not associated with increased total plasma homocysteine. No additional effect of A1298C on total plasma homocysteine was observed in 148 combined heterozygotes compared with 98 heterozygotes for the C677T alone. These findings do not support a major role for the A1298C mutation in homocysteine metabolism and emphasize the hypothesis that MTHFR genotypes may interfere with coronary artery disease risk only when an unbalanced nutritional status leads to raised total plasma homocysteine levels.

Interaction between smoking and PON2 Ser311Cys polymorphism as a determinant of the risk of myocardial infarction

Background  Increased oxidative stress is thought to play a role in the pathogenesis of the atherothrombotic process. Paraoxonases (PONs) are closely related antioxidant enzymes encoded by clustered genes on chromosome 7q. We evaluated three PON polymorphisms (PON1 Leu55Met and Gln192Arg; PON2 Ser311Cys) as possible risk factors for coronary atherosclerotic disease (CAD) and/or its main thrombotic complication, myocardial infarction (MI).

Enhanced Levels of Oxidized Low-Density Lipoprotein Prime Monocytes to Cytokine Overproduction via Upregulation of CD14 and Toll-Like Receptor 4 in Unstable Angina

Objectives—The purpose of this study was to establish whether oxidized low-density lipoprotein (oxLDL) contributes to cytokine overproduction via upregulation of CD14 and toll-like receptor-4 (TLR-4) expression on circulating monocytes of unstable angina (UA) patients. Methods and Results—Expression of CD14 and TLR-4 on circulating monocytes, and the concentration of plasma oxLDL, (interleukin [IL])-6, IL-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) were measured in 27 control (C) subjects, 29 patients with stable angina (SA), and 27 with UA. CD14 and TLR-4 expression on monocytes and circulating IL-6, IL-1 beta, and oxLDL were higher in UA than in SA and C subjects (P<0.001). In in vitro experiments, oxLDL increased CD14 and TLR-4 expression (P<0.001) in control monocytes as well as IL-6, IL-1 beta, and at a lower extent TNF-&agr; and MCP-1 levels in the supernatant (P from <0.05 to <0.001). The preincubation of sera derived from UA patients but with control monocytes also induced a significant increase of CD14 and TLR-4 expression (P<0.001) and of IL-6 and IL-1 beta production (P<0.001) in the supernatant. Conclusions—In UA patients oxLDL may contribute to monocyte overproduction of some cytokines by upregulating CD14 and TLR-4 expression.

Expansion of necrotic core and shedding of Mertk receptor in human carotid plaques: a role for oxidized polyunsaturated fatty acids?

AIMS Expansion of necrotic core (NC), a major feature responsible for plaque disruption, is likely the consequence of accelerated macrophage apoptosis coupled with defective phagocytic clearance (efferocytosis). The cleavage of the extracellular domain of Mer tyrosine kinase (Mertk) by metallopeptidase domain17 (Adam17) has been shown to produce a soluble Mertk protein (sMer), which can inhibit efferocytosis. Herein, we analysed the expression and localization of Mertk and Adam17 in the tissue around the necrotic core (TANC) and in the periphery (P) of human carotid plaques. Then we studied the mechanisms of NC expansion by evaluating which components of TANC induce Adam17 and the related cleavage of the extracellular domain of Mertk. METHODS AND RESULTS We studied 97 human carotid plaques. The expression of Mertk and Adam17 was found to be higher in TANC than in P (P < 0.001). By immunohistochemistry, Mertk was higher than Adam17 in the area of TANC near to the lumen (P < 0.01) but much lower in the area close to NC (P < 0.01). The extract of this portion of TANC increased the expression (mRNA) of Adam17 and Mertk (P < 0.01) in macrophage-like THP-1 cells but it also induced the cleavage of the extracellular domain of Mertk, generating sMer in the medium (P < 0.01). This effect of TANC extract was most evoked by its content in F(2)-isoprostanes, hydroxyoctadecadienoic acids, and hydroxytetraenoic acids. CONCLUSION Some oxidized derivatives of polyunsaturated fatty acids contained in TANC of human carotid plaques are strong inducers of Adam17, which in turn leads to the generation of sMer, which can inhibit efferocytosis.

Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs) following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L) for 24 hours and oxidative stress was induced by the addition of oxidized (ox)-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin); proteins linked to inflammation (IL-6 and TNFalpha), thrombotic state (tissue factor, PAI-1 and uPA), hypertension such as endothelin-1 (ET-1), and vascular remodeling such as metalloproteinases (MMP-2, MMP-9) and protease inhibitor (TIMP-1). The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.