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.

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.

Are antiphospholipid antibodies an independent risk factor for atherosclerosis?

The purpose of this study was to check whether antiphospholipid antibodies (aPL) could be an independent risk factor for atherosclerosis. Eighty-five subjects were studied: 45 with primitive antiphospholipid antibody syndrome and 40 controls affected by deep vein thrombosis secondary to known causes. The two groups were homogeneous for age, sex, and risk factors for atherosclerosis. All the subjects submitted to echo-color doppler of the carotid arteries, femoral arteries, and abdominal aorta. The cases were then subdivided into three subgroups on the basis of the positivity to the three subpopulations of aPL. Results demonstrate that there is no correlation between aPL and atherosclerosis. The different positivity to aPL does not modify this conclusion.

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.

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.

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.