The atherosclerotic plaque vulnerability: focus on the oxidative and endoplasmic reticulum stress in orchestrating the macrophage apoptosis in the formation of the necrotic core.

Although the understanding the pathophysiology of atherogenesis and atherosclerosis progression has been one of the major goals of cardiovascular research during the last decades, the precise mechanisms underlying plaque destabilization are still unknown. The disruption of the plaque and the thrombosis in the lumen that are mostly determined by the expansion of the necrotic core (NC) are driven by various mechanisms, including accelerated macrophage apoptosis and defective phagocytic clearance (defective efferocytosis). Oxidative stress is implicated in the expansion of the NC: in fact, many oxidized compounds and processes contribute to the macrophage apoptosis; in addition, the oxidized derivatives of polyunsatured fatty acids promote defective efferocytosis, with the final result of NC expansion. In the last years the role of the endoplasmic reticulum (ER) stress is under investigation to better define its possible contribution in affecting the NC expansion. The abnormal amount of apoptotic cells in the vulnerable plaque has been demonstrated to be related both to the sustained ER stress and to the expression of survival and protective genes, such as the unfolded protein response or/and the nuclear erytroid- related factor 2. In this review the authors focus on the promising results of the oxidative and ER stress in contributing to triggering and orchestrating the atherosclerotic plaque vulnerability.

Role of MDCT coronary angiography in the clinical setting: economic implications.

PURPOSE: This study evaluated the incremental value and cost-effectiveness ratio of introducing coronary angiography (CA) with multidetector computed tomography (MDCT-CA) in the diagnostic management of patients with suspected coronary artery disease (CAD) compared with the traditional diagnostic workup. MATERIAL AND METHODS: Five hundred and fifty consecutive patients who underwent MDCT-CA between January 2009 and June 2011 were considered. Patients with atypical chest pain and suspected obstructive CAD were directed to one of two diagnostic pathways: the traditional protocol (examination, stress test, CA) and the current protocol (examination, stress test, MDCT-CA, and CA, if necessary). The costs of each protocol and for the individual method were calculated. Based on the results, the cost-effectiveness ratio of the two diagnostic pathways was compared. A third, modified, diagnostic pathway has been proposed with its relative cost-effectiveness ratio (examination, MDCT-CA, stress test, and CA, if necessary). RESULTS: Stress test vs. MDCT-CA had an accuracy of 66%, a sensitivity and specificity of 21% and 87%, respectively, and a positive (PPV) and negative (NPV) predictive value of 40% and 70%, respectively. Comparison between conventional CA (CCA) and MDCT-CA showed a sensitivity and specificity of 92% and 89%, respectively, a PPV and NPV of 89%, and an accuracy of 92%. The traditional protocol has higher costs than the second protocol: 1,645 euro against 322 euro (mean), but it shows a better cost-effectiveness ratio. The new proposed protocol has lower costs, mean 261 euro, with a better costeffectiveness ratio than the traditional protocol. CONCLUSIONS: The diagnostic protocol for patients with suspected CAD has been modified by the introduction of MDCT-CA. Our study confirms the greater diagnostic performance of MDCT-CA compared with stress test and its similar accuracy to CCA. The use of MDCT-CA to select patients for CCA has a favourable cost-effectiveness profile.

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.

Nebivolol reduces asymmetric dimethylarginine in endothelial cells by increasing dimethylarginine dimethylaminohydrolase 2 (DDAH2) expression and activity.

Asymmetric dimethylarginine (ADMA) has been reported to affect the synthesis of nitric oxide (NO) in endothelial cells by inhibiting endothelial NO synthase (eNOS) activity and to cause endothelial dysfunction in humans. This study was conducted in human umbilical vein endothelial cells (HUVECs) to evaluate the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on ADMA concentration and on dimethylarginine dimethylaminohydrolase (DDAH2), the enzyme that regulates ADMA catabolism. Nebivolol dose-dependently decreased ADMA/symmetric dimethylarginine (SDMA) ratio (p from <0.01 to <0.001). This was parallelled by a dose-dependent increase in DDAH2 mRNA (p from <0.01 to <0.001) and protein expression (p from <0.01 to <0.001) and activity (p from <0.01 to <0.001). The small interference RNA (siRNA)-mediated knockdown of DDAH2 abolished the modification of DDAH2 expression (p<0.001) and ADMA/SDMA ratio (p<0.001) induced by nebivolol. In conclusion, the results of this study demonstrate that nebivolol reduces ADMA concentration by increasing DDAH2 expression and activity. Our in vitro findings describe a novel vascular effect of nebivolol and clearly identify this compound as the first antihypertensive agent that modulates DDAH2 in endothelial cells.

Effect of sulfhydryl and non-sulfhydryl angiotensin-converting enzyme inhibitors on endothelial function in essential hypertensive patients.

BACKGROUND: Oxidative inactivation of nitric oxide (NO) is regarded as an important cause of reduced endothelium-dependent vasodilation in essential hypertension. Because zofenopril, an angiotensin-converting enzyme (ACE) inhibitor with a sulfhydryl (SH) group, has demonstrated antioxidant properties and to reduce adhesion molecule expression in vitro, in this study we evaluated the effect of this drug in comparison with the carboxylic ACE inhibitor ramipril and the beta-adrenoreceptor blocker atenolol on (1) circulating adhesion molecules and some oxidative stress parameters and (2) endothelium-dependent vasodilation in essential mildly hypertensive patients. METHODS: A total of 45 healthy subjects and 45 matched hypertensive patients participated in the study. Hypertensive patients were randomly treated with zofenopril (15 to 30 mg/d), ramipril (2.5 to 5 mg/d), and atenolol (50 to 100 mg/d). At baseline and after an 8-week therapy we evaluated blood pressure (BP) values, plasma and LDL hydroperoxides, plasma 8-isoprostanes, circulating levels of oxidized-(ox)LDL and of adhesion molecules (intercellular cell adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1], and E-selectin). Furthermore, all patients underwent ultrasound detection of brachial artery reactivity and endothelium-dependent dilation (flow-mediated dilation, FMD) was evaluated. RESULTS: All the treatments determined similar significant (P < .001) reduction of both systolic and diastolic BP values. Plasma (P < .01) and LDL hydroperoxides (P < .01), plasma 8-isoprostanes (P < .05), circulating oxLDL (P < .05), and adhesion molecules (P < .05) were significantly reduced only in patients receiving zofenopril. Similarly FMD was significantly increased (P < .001) in the zofenopril-treated group. CONCLUSIONS: Our results suggest that in mildly hypertensive patients without organ damage zofenopril, beyond its BP-lowering effects and through its sustained antioxidant activity, offers important advantages in reducing endothelial activation.

Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells.

Nebivolol, a third generation selective beta(1)-adrenoceptor (beta(1)-AR) antagonist, has been reported to reduce intracellular oxidative stress and to induce the release of nitric oxide (NO) from the endothelium. Nebivolol is also subjected to a complex metabolic process where glucuronidation, aromatic and alicyclic hydroxylation are the major pathways leading to several metabolites. We have studied the effect of nebivolol, its enantiomers and metabolites on intracellular oxidative stress and NO availability in human umbilical vein endothelial cells (HUVECs). Furthermore, since the receptors involved in this endothelial effect of nebivolol remain controversial, we have studied this matter by the use of antagonists of beta-AR. dl-Nebivolol, d-nebivolol and l-nebivolol significantly reduced the formation of reactive oxygen species (ROS) and superoxide induced by oxidized-low density lipoprotein (ox-LDL), although the racemic and l-form were significantly more active than d-nebivolol in this activity. A marked decrease in the availability of intracellular NO was found in HUVECs exposed to ox-LDL and this parameter was normalized by the prior incubation with dl-nebivolol, d-nebivolol and l-nebivolol; the effect of racemate was mainly mimicked by its l-enantiomer. eNOS activity significantly increased by a 5-min contact of HUVECs with dl-nebivolol and l-nebivolol, but not with d-nebivolol, and a similar pattern was observed when the intracellular calcium increase was measured. The metabolites A2, A3', A12 and A14 but not A1, A3 and R 81,928, derived from different metabolic pathways, retained the antioxidant activity of the parent racemic compound dl-nebivolol, reducing the intracellular formation of ROS and superoxide. The effects of dl-nebivolol on intracellular formation of NO, eNOS activity and intracellular Ca(2+) were partially antagonized by the antagonists of beta(1-2)-AR nadolol or by the beta(3)-AR antagonist SR59230A and further antagonized by their combination or by (beta(1-2-3)-AR antagonist bupranolol. In conclusion, this study shows that the NO releasing effect of nebivolol is mainly due to its l-enantiomer; the racemate and its enantiomers possess a remarkable antioxidant activity that contributes to its effect on the cellular NO metabolism and the activation of beta(3)-AR through a calcium dependent pathway is involved in the mechanisms leading to the NO release.

Plasma levels of oxidized-low-density lipoproteins are higher in patients with unstable angina and correlated with angiographic coronary complex plaques.

OBJECTIVES: To measure circulating levels of oxidized-low-density lipoproteins (ox-LDL) in patients with stable and unstable angina and controls, and to investigate their correlation with the extent of coronary artery disease (CAD) and the presence of complex plaques at coronary angiography. METHODS AND RESULTS: Circulating ox-LDL were assessed, using ELISA, in patients with unstable angina (UA, n=26), stable angina (SA, n=29) and in controls (C, n=27). All patients underwent coronary angiography. The extent of CAD was evaluated using a quantitative score, while the presence of complex, vulnerable plaques was angiographically assessed. Ox-LDL were higher in UA patients than in SA patients and in C subjects, and in SA patients than in C subjects (C, 45.6+/-12.8 U/L; SA, 58.8+/-11.0 U/L; UA, 73.7+/-13.6 U/L; p<0.001). No correlation was found with the extent of atherosclerotic disease in the coronary tree. Patients with angiographic complex lesions showed significantly higher levels of ox-LDL (68.4+/-13.9 U/L versus 55.2+/-16.4 U/L, p<0.001). Multiple regression analysis showed that ox-LDL were independent predictors of the presence of complex plaques (p<0.023). CONCLUSIONS: Ox-LDL levels are higher in unstable patients and correlate with the presence of angiographically documented complex plaques. Ox-LDL might be markers of destabilization of CAD.

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice treated with lacidipine is associated with a decreased susceptibility of low-density lipoprotein to oxidation.

A study has been carried out in the apolipoprotein (apo) E-deficient mouse to investigate the activity of lacidipine (a calcium antagonist with antioxidant properties) in inhibiting the development of atherosclerotic lesions; of particular interest were changes in the susceptibility of low-density lipoproteins (LDL) to oxidation. Mice receiving a Western-type diet to accelerate the development of atherosclerosis were treated orally with vehicle or lacidipine at 3 or 10 mg/kg/day for 8 weeks. Lacidipine treatment (at 3 or 10 mg/kg) had no effect on the plasma lipid profile. However, a significant (P < 0.01) dose-related reduction of 43 and 50% of the aortic lesion area in respect to vehicle-treated mice was observed. Moreover, the resistance of mouse plasma LDL to undergo lipid peroxidation was significantly (P < 0.01) increased in apo E-deficient mice treated with lacidipine. The native LDL-like particle, derived from apo E-deficient mice treated with lacidipine, contained significantly lower concentrations of malonyldialdehyde than the vehicle-treated control group (P < 0.01). After exposure to human umbilical vein endothelial cells, LDL-like particle vitamin E levels (expressed as area under the curve; AUC), were significantly higher (P < 0.01) in both the 3 and 10 mg/kg lacidipine-treated groups, in comparison with the vehicle-treated control animals. We conclude that lacidipine reduced the extent of the atherosclerotic area in hypercholesterolaemic apo E-deficient mice, and that this reduction may be associated with the capacity of the drug to decrease the susceptibility of LDL to oxidation.

Beta2 integrin-dependent neutrophil adhesion induced by minimally modified low-density lipoproteins is mainly mediated by F2-isoprostanes.

BACKGROUND: Oxidation of LDL produces a series of biologically active, oxidized lipids. Among them, isoprostanes, and in particular iPF(2alpha)-III, seem to be crucial in mediating some of the key cellular events seen in myocardial ischemia-reperfusion injury. METHODS AND RESULTS: Minimally modified LDL (MM-LDL) triggers a dose-dependent, very rapid neutrophil adhesion to human fibrinogen. Rapid adhesion triggering correlates with degree of LDL oxidation and accumulation of isoprostanes. Isoprostanes accumulated in MM-LDL are major determinants of the proadhesive effect of oxidized LDL, as shown by experiments of receptor functional deletion. Moreover, evidence is provided of expression on human neutrophils of a biological active isoprostane receptor distinct from the classical thromboxane A2 receptor. CONCLUSIONS: These data suggest that isoprostanes are major contributors to the proadhesive effect induced by MM-LDL on neutrophils and provide additional evidence for the involvement of isoprostanes in the pathogenesis of myocardial ischemia/reperfusion injury.