Author + information
- Received December 31, 2002
- Revision received June 6, 2003
- Accepted June 10, 2003
- Published online November 19, 2003.
- Luciano Cominacini, MD*,* (, )
- Anna Fratta Pasini, MD*,
- Ulisse Garbin, MD*,
- Cristina Nava, MD*,
- Anna Davoli, BSc*,
- Marco Criscuoli, BSc†,
- Attilio Crea, BSc‡,
- Tatsuya Sawamura, MD§ and
- Vincenzo Lo Cascio, MD*
- ↵*Reprint requests and correspondence:
Dr. Luciano Cominacini, Dipartimento di Scienze Biomediche e Chirurgiche (Medicina D), Università di Verona, Ospedale Policlinico, 37134 Verona, Italy.
Objectives The objective of the present study was to elucidate the vasodilator mechanisms of nebivolol, a high selective β1-receptor antagonist with antioxidant properties.
Background Oxidative inactivation of nitric oxide (NO) is regarded as an important cause of its decreased biological activity.
Methods Oxidative stress was induced through the binding of oxidized (ox)-low-density lipoprotein (LDL) to its specific endothelial receptor, called “lectin-like oxidized LDL receptor-1” (LOX-1), in bovine and human endothelial cells and in Chinese hamster ovary cells stably expressing bovine LOX-1 (BLOX-1-CHO cells). Reactive oxygen species (ROS), superoxide (O2·−), and NO were measured in cells by flow cytometry.
Results Nebivolol and its 4-keto derivative prevented in a dose-dependent manner the increase of ROS (p < 0.001) and O2·−(p < 0.001) in bovine aortic endothelial cells (BAECs), human umbilical vein endothelial cells (HUVECs), and BLOX-1-CHO cells stimulated with ox-LDL. Atenolol had no effect. The incubation of HUVECs and BAECs with ox-LDL reduced basal and bradykinin-induced NO and nitrite concentration (p from <0.001 to <0.01). Nebivolol and its 4-keto derivative prevented the reduction of basal and stimulated NO and nitrite concentration (p from <0.001 to <0.01) while atenolol had no effect. The preincubation of BAECs with blocking anti-LOX-1 monoclonal antibody (LOX-1 mAb) significantly counteracted the effect of ox-LDL on stimulated generation of NO (p < 0.001), but the effect was significantly lower than that of nebivolol and its 4-keto derivative alone (p < 0.01).
Conclusions In conclusion, the findings of the present study indicate that nebivolol increases NO also by decreasing its oxidative inactivation.
Endothelium plays an important role in the regulation of vascular tone, the major endothelium-derived relaxing factor being nitric oxide (NO) (1,2). It is recognized that essential hypertension is characterized by impaired endothelium-dependent vasodilation (3), an abnormality that is known to precede atherosclerosis and particularly coronary artery disease (CAD) (4). This is especially relevant because hypertension is a major risk factor for CAD.
The impairment of endothelium-dependent relaxation has been linked to a decreased production and/or biological activity of endothelium-derived NO (5).
Nebivolol, a highly selective β1-receptor antagonist with antioxidant properties (6,7), has been shown to cause vasodilation in animal models (8)and humans (9)and to reverse endothelial dysfunction in essential hypertension (10). It has been hypothesized that nebivolol induces vasodilation through an endothelial β2-adrenergic receptor-mediated NO production (11).
Because the vascular release of superoxide (O2·−) is increased in atherosclerotic arteries (12)and O2·−can inactivate NO in a chemical reaction during which peroxynitrite is formed (13), oxidative inactivation of NO has also been regarded as an important cause of endothelial dysfunction in essential hypertension (14).
The present study was therefore performed to obtain further insight into the mechanism underlying the effect of nebivolol on NO production and, in particular, to explore whether nebivolol reduces oxidative inactivation of NO and, consequently, increases its intracellular availability in endothelial cells exposed to oxidative stress.
Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described (15,16). Chinese hamster ovary-K1 (CHO-K1) cells and a CHO-K1 cell line stably expressing bovine lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (BLOX-1-CHO) (17)were cultured as previously described (15). Cell survival was monitored according to the method of Landegren (18).
Addition and measurement of nebivolol in endothelial cells
Nebivolol and its 4-keto and 4-OH derivatives (courtesy of Menarini Ricerche, Firenze, Italy) and atenolol (Sigma, St. Louis, Missouri) were dissolved in ethanol and in M-199. Increasing amounts of the drugs (from 1 to 25 μmol/l) were added to endothelial cell monolayers for 30 min at 37°C. Nebivolol was also incubated at different times (30 to 180 min) with endothelial cells in order to ascertain whether endothelial cells metabolize the drug. The determination of nebivolol and atenolol in cells was performed as previously described (19,20).
Induction of oxidative stress in endothelial cells
Oxidative stress in endothelial cells was induced by the addition of oxidized (ox) LDL. The binding of ox-LDL to the endothelial receptor called LOX-1 has already been demonstrated to increase the intracellular concentration of reactive oxygen species (ROS) (15)and O2·−(21).
LDL isolation and oxidation
Whole blood, containing ethylenediamine tetraacetic acid (EDTA) (1 mg/ml), was processed for LDL separation as previously described (15). Cu2+modified LDL was prepared as reported (15), and the extent of LDL oxidation was determined by thiobarbituric acid-reactive substances (TBARS) (15). Protein was measured by the Pierce BCA protein assay reagent (22).
Translocation of cytosolic nicotinamide adenine dinucleotide phosphate oxidase components
The translocation of p47phox and Rac1 was evaluated as previously described (23). Briefly, HUVECs (5 × 107), incubated with 50 μg protein/ml of ox-LDL and native LDL (control) for 1 to 10 min at 37°C, were sonicated on ice in 0.5 ml relaxation buffer (10 mM Pipes, pH 7.4, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin). Cytosol and membranes were prepared by centrifugation on a discontinuous gradient of 15% and 34% (w/w) sucrose at 120.000 gfor 30 min at 4°C. The membrane pellets were transferred to a nitrocellulose membrane and probed with the anti-p47phox (Santa Cruz Biotechnology, Santa Cruz, California) or anti-Rac1 antibody (Santa Cruz Biotechnology).
Before stimulation with ox-LDL, HUVECs were also preincubated with anti-human LOX-1 monoclonal antibody (hLOX-1 mAb, 10 μg/ml), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (Calbiochem, La Jolla, California) (10 μmol/l), protein kinase C (PKC) inhibitor bisindolylmaleimide (Calbiochem) (1 μmol/l), and increasing amounts of nebivolol (from 5 to 25 μmol/l) for 30 min at 4°C.
ROS and O2·−measurement
Intracellular ROS and O2·−levels were measured as described (15,21)by following the oxidation of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE) by flow cytometry (Coulter Corp., Hialeah, Florida). Cells were also tested for the production of H2O2(24)and extracellular O2·−(25)radicals induced by ox-LDL.
For ROS, H2O2, and O2·−measurements, endothelial cells were first preincubated with nebivolol or atenolol for 30 min at 37°C, then increasing concentrations (50 to 150 μg protein/ml) of ox-LDL and native LDL were incubated with the cells for 10 min at 37°C in the presence of 5 mmol/l arginine and 3 μmol/l tetrahydrobiopterin (TB4). The effect of 4-OH, and 4-keto derivatives of nebivolol on the intracellular ROS and O2·−concentrations was also evaluated under the previously specified experimental conditions.
To test the response specificity, nebivolol, its 4-keto derivative, and anti-bovine LOX-1 monoclonal antibody (bLOX-1 mAb) (30 μg/ml) (17)were incubated with BAECs, CHO-K1, and BLOX-1-CHO cells under the previously specified experimental conditions.
Binding of labeled ox-LDL to BAECs and bLOX-1-CHO cells and inhibition of binding
Oxidized LDL was labeled using the fluorescent compound N, N-dipentadecyl-aminostyrylpyridinium iodide (di-15-ASP) as described previously (15). The data were fitted with GraFit (Erithacus Software Ltd., Staines, United Kingdom). A 100-fold excess of unlabeled ox-LDL, native LDL (100 μg protein/ml), bLOX-1 mAb (30 μg/ml), polyinosinic acid (250 μg/ml), and nebivolol (25 μmol/l) were used as inhibitors.
Intracellular NO was measured in the presence and absence of endothelial activation with bradykinin (100 nmol/l) (Sigma), as previously described, (21)by utilizing the fluorescent indicator 4, 5 diaminofluorescein diacetate (DAF-2 DA) in flow cytometry (Coulter Corp.). Nitric oxide production was also monitored by following levels of nitrite in the supernatant of stimulated HUVECs and BAECs. Levels of nitrite were determined by a fluorimetric assay (26). For NO measurement, HUVECs and BAECs were first preincubated with nebivolol or atenolol for 30 min at 37°C, then increasing concentrations (50 to 150 μg protein/ml) of ox-LDL and native LDL were incubated with endothelial cells for 10 min at 37°C in the presence of bradykinin (100 nmol/l), 5 mmol/l arginine, and 3 μmol/l TB4. The effect of 4-OH and 4-keto derivatives of nebivolol on the intracellular basal and stimulated NO concentration was also evaluated under the previously specified experimental conditions.
In order to ascertain whether the effect of ox-LDL on intracellular NO concentration was dependent on O2.-produced by the binding of ox-LDL to LOX-1, bLOX-1 mAb (17)(30 μg/ml) was used under the previously specified experimental conditions.
Furthermore, to test the contribution of endothelial α- or β-adrenergic receptors and 5-HT serotonergic receptors to NO concentration after preincubation with nebivolol and metabolites, a β2-adrenergic antagonist (butoxamine, 0.1 mmol/l; Sigma), an α-adrenergic receptor blocker (phentolamine 10 μmol/l; Sigma), or a 5-HT antagonist (NAN-190; 1 μmol/l; ICN, Frankurt/Main, Germany) was used.
Endothelial NO synthase activity measurement
Endothelial NO synthase (eNOS) activity was measured by evaluating the metabolism of 3H arginine to 3H citrulline as described previously (21).
Statistical analysis was performed by one-way analysis of variance with repeated measures followed by post-hoc Tukey's test for multiple comparisons using the “SYSTAT” program and statistical software manual (SYSTAT Inc., Evanston, Illinois) for Macintosh. Statistical significance was inferred when p < 0.05. All reported p values were derived from Tukey's test.
The addition of increasing amounts of nebivolol and atenolol in HUVECs and BAECs determined a dose-dependent intracellular increase of the drugs. The intracellular recovery was higher for nebivolol than for atenolol (respectively ranging from 65 ± 6% to 70 ± 8% for nebivolol and metabolites and of 35 ± 4% for atenolol (p < 0.001). The concentration of the nebivolol in endothelial cells was stable for at least 3 h.
In our experimental conditions, the incubation of HUVECs and BAECs with increasing amounts of ox-LDL for 10 min at 37°C induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·−(p from <0.001 to <0.01, in experiments performed in triplicate on six different occasions for each concentration of ox-LDL). Similarly, levels of H2O2and O2·−in the supernatant of both HUVECs and BAECs significantly increased after the addition of ox-LDL (p from <0.001 to <0.01).
To determine whether the increase of ROS and O2·−induced by ox-LDL was dependent on NADPH oxidase activation and was, therefore, preceded by the translocation of some cytosolic NADPH oxidase components, we isolated membrane from ox-LDL-stimulated HUVECs and carried out immunoblot analysis of p47phox and Rac1. The translocation of p47phox and Rac1 was first found at 1 min after stimulation and peaked at 4 min (data not shown). We found that incubation of ox-LDL with HUVECs determined a significant increase in both p47phox and Rac1 translocation. The translocation of p47phox and Rac1 was inhibited by hLOX-1 mAb and respectively by bisindolylmaleimide and SB203580. Figure 1shows representative immunoblots of p47phox and Rac1 and the summarized data obtained by densitometric analysis. Nebivolol and its 4-keto derivative dose-dependently reduced the intracellular concentration of ROS and O2·−in HUVECs and BAECs (p from <0.001 to <0.01). Atenolol and 4-OH derivative of nebivolol had no effect. Figure 2Ashows the effect of nebivolol, its 4-keto derivative, and atenolol on O2·−-induced by ox-LDL in HUVECs. The effect of nebivolol on intracellular O2·−concentration was associated to a reduced translocation of both p47phox and Rac1; the effect, however, was much more evident for p47phox (Fig. 2B).
Nebivolol (25 μmol/l) also reduced H2O2from 4.52 ± 0.25 nmol/106cells to 2.1 ± 0.21 nmol/106cells in HUVECs (p < 0.001) and from 4.92 ± 0.44 nmol/106 cells to 1.99 ± 0.24 nmol/106cells in BAECs (p < 0.001); it also decreased supernatant O2·−from 10.1 ± 0.8 nmol/106cells to 4.6 ± 0.5 nmol/106cells in HUVECs (p < 0.001) and from 9.6 ± 0.9 nmol/106cells to 4.2 ± 0.5 nmol/106cells in BAECs (p < 0.001). Atenolol had no effect. All the experiments regarding H2O2and O2·−in the supernatant were performed in triplicate on six different occasions.
To test the response specificity of nebivolol and its 4-keto derivative, a fixed amount of ox-LDL (50 μg protein/ml) was incubated with BAECs, CHO-K1, and BLOX-1-CHO cells for 10 min at 37°C after preincubation with bLOX-1 mAb, nebivolol, and its 4-keto derivative for 30 min at 37°C. As shown in Figure 3, the O2·−concentration was markedly reduced in BAECs and BLOX-1-CHO cells preincubated with bLOX-1 mAb (p < 0.001), nebivolol (p < 0.001), and its 4-keto derivative (p < 0.001).
To exclude an interference of nebivolol on ox-LDL binding to LOX-1, binding tests with labeled ox-LDL were performed in BAECs and BLOX-1-CHO cells. The dose-response relationship in di-15-ASP-labeled ox-LDL binding to BAECs and BLOX-1-CHO cells showed saturation kinetics, and a 100-fold excess of unlabeled ox-LDL completely inhibited di-15-ASP-labeled ox-LDL binding to BAECs and BLOX-1-CHO cells (data not shown). Scatchard analysis revealed that di-15-ASP-labeled ox-LDL can bind to BAECs and BLOX-1-CHO cells with a constant dissociation ranging from 31 to 37 μg/ml. Oxidized LDL, bLOX-1 mAb, and polyinosinic acid significantly reduced the binding of di-15-ASP-labeled ox-LDL both in BAECs and BLOX-1-CHO cells (p from <0.001 to <0.01). Nebivolol, its 4-keto derivative, and native LDL had no effect.
The incubation of HUVECs and BAECs with ox-LDL for 10 min in presence of DAF-2 DA, dose-dependently reduced basal and bradykinin-induced intracellular NO concentration (p from <0.001 to <0.01, n = 12 for each concentration of ox-LDL). Nebivolol and its 4-keto derivative prevented the reduction of basal and stimulated intracellular NO induced by a fixed dose of ox-LDL (50 μg protein/ml) in HUVECs and BAECs (p from <0.001 to <0.01) while atenolol did not. Figure 4shows the effect of nebivolol, its 4-keto derivative, and atenolol on the reduction of basal and stimulated intracellular NO concentration induced by ox-LDL in BAECs. Butoxamine, a β2-adrenergic antagonist; phentolamine, an α-adrenergic receptor blocker; and NAN 190, a 5-HT receptor antagonist had no effect on the counteracting effect of nebivolol.
Similarly, levels of nitrite in the supernatant of bradykinin-stimulated endothelial cells dropped from 635 ± 25 pmol/well to 211 ± 12 pmol/well (p < 0.001, n = 12) in HUVECs and from 696 ± 45 pmol/well to 186 ± 18 pmol/well in BAECs (p < 0.001, n = 12) after the addition of 50 μg protein/ml of ox-LDL. The decrease was prevented by nebivolol (25 μmol/l) (respectively from 642 ± 32 pmol/well to 612 ± 38 pmol/well in HUVECs, and from 711 ± 54 pmol/well to 677 ± 44 pmol/well in BAECs, p = not significant, n = 12) but not by atenolol at the same concentration.
To test if the restoring action of nebivolol on the reduction of intracellular NO concentration induced by ox-LDL was due only to its effect on O2·−, we preincubated BAECs with bLOX-1 mAb and measured bradykinin-stimulated intracellular NO after induction of oxidative stress with ox-LDL. Figure 5shows that the preincubation of BAECs with bLOX-1 mAb significantly counteracted the effect of ox-LDL on bradykinin-stimulated generation of NO (p < 0.001). The counteracting effect of bLOX-1 mAb on intracellular NO was significantly lower than that induced by nebivolol and its 4-keto derivative alone (p < 0.01) or nebivolol or its 4-keto derivative plus bLOX-1 mAb (p < 0.001).
The results of this study also show that the eNOS activity of BAECs significantly increased by a 5-min contact with 25 μmol/l nebivolol; this effect was almost identical to that induced by 100 nmol/l bradykinin (Fig. 6). Atenolol and ox-LDL had no effect on eNOS activity.
The results of this study show that nebivolol and its 4-keto derivative reduce the concentration of ROS and, in particular, of O2·−induced by ox-LDL in endothelial cells. The increase in ROS and O2·−was previously shown to be specifically related to the binding of ox-LDL to LOX-1 and to be almost totally prevented by radical scavengers and by bLOX-1 mAb (15,21). The fact that in this study nebivolol and its 4-keto derivative work not only in endothelial cells, but also in BLOX-1-CHO cells suggests that the effect of the two compounds is specific for the ROS produced by the binding of ox-LDL to LOX-1. This, of course, does not mean that LOX-1 is the only receptor involved in nebivolol-dependent effect because any other receptor producing ROS or O2·−after stimulation (27,28)could be a potential target of nebivolol activity. Nebivolol has already been demonstrated to possess antioxidant activity (6,7). The effect of nebivolol and, above all, of its 4-keto derivative we found in this study may, therefore, be related to their radical scavenging activity. The possibility that nebivolol can affect the binding of ox-LDL to LOX-1 is excluded by the binding tests with labeled ox-LDL.
Furthermore, it has been previously hypothesized that endothelial NADPH oxidase may be the source of the free radicals generated by activated LOX-1 (21,29). NADPH oxidase is a multicomponent enzyme consisting of at least two membrane components (gp91phox and p22phox) and three cytosolic proteins (p47phox, p67phox, and Rac) (30). All the classic NADPH subunits, including p47phox and Rac1 have already been shown to be expressed in human endothelial cells and in particular in HUVECs (31). In resting cells the oxidase is dormant and its components separately exist between the membrane and the cytosol (30). When cells are exposed to appropriate stimuli, NADPH oxidase is activated by association of these components on the plasma membrane (30). Actually, neutrophil and endothelial NADPH oxidase exhibit differences in their activity. In fact, a substantial proportion of NADPH oxidase in unstimulated endothelial cells, in contrast to the neutrophil enzyme, exists as a preassembled intracellular complex associated with the cytoskeleton (31). Nevertheless, a significant translocation of p47phox to the plasma membrane was demonstrated under stimulation with angiotensin II (32), consistent with the oxidase being present in the membrane fraction. Taken together, these findings support the conclusion of our study that the translocation of p47phox and Rac1 really occurred in our experimental conditions and that endothelial NADPH oxidase may be the source of the free radicals generated by activated LOX-1. The fact that the translocation of p47phox and Rac1 was greatly reduced, respectively, by bisindolylmaleimeide and SB203580 may indicate that p38 MAPK is more responsible for the translocation of Rac and PKC for the translocation of p47phox. In this context, it is of interest that these two kinases have already been shown to be associated with LOX-1 activation (33,34). Because in this study the translocation of Rac1 and in particular of p47phox was reduced by nebivolol, a conclusion can be drawn that this compound and its 4-keto derivative may reduce ROS production also by affecting the signaling pathways leading to NADPH oxidase activation.
Previous findings by our group have shown that the binding of ox-LDL (21)and platelets (29)to LOX-1 reduces the intracellular and extracellular concentration of NO in endothelial cells through an increased cellular production of O2·−. In the present study, we demonstrated that nebivolol and its 4-keto derivative prevented the reduction of basal and bradykinin-stimulated NO induced by ox-LDL in BAECs. Because the reduction of NO was counteracted by bLOX-1 mAb, another conclusion of this study is that the effect of nebivolol can be at least partially explained by its effect on O2·−, which can inactivate NO in a chemical reaction during which peroxynitrite is formed (13). From our results it is, however, clear that the effect of nebivolol and its 4-keto derivative on NO cannot be explained only by their effect on O2·−because the counteracting effect of bLOX-1 mAb was significantly lower than that caused by nebivolol and its 4-keto derivative alone or in combination with the LOX-1 blocking antibody. Taken together, these results suggest that nebivolol and its 4-keto-derivative may increase NO in endothelial cells by working in at least two different ways: they reduce O2·−concentration and consequently peroxynitrite formation and increase NO production. The conclusion on the effect of nebivolol on NO production is supported by the results we obtained by measuring eNOS activity in endothelial cells. The activity of the enzyme was more than doubled by a 5-min contact with nebivolol; this effect was identical with that induced by bradykinin. It is noteworthy that atenolol and ox-LDL were devoid of any effect on eNOS activity. Our results on NO production are in agreement with a series of previous studies showing that the mechanism of vasodilator action of nebivolol is attributed to activation of eNOS in vascular endothelial cells (9,11,35).
The results of this study and, in particular, the inhibitory effect of nebivolol on O2·−concentration may have implications in vivo. In fact, oxidative inactivation of NO has been regarded as an important cause of endothelial dysfunction in essential hypertension (14). Furthermore, it has been recognized for a long time that atherosclerotic blood vessels are very susceptible to the development of vasospasm in vivo (36), and the current weight of evidence suggests that impaired endothelium-dependent vasodilation plays a central role in the mechanisms leading to ischemic manifestations (37). In this context, it has been demonstrated that peroxynitrite, generated by a rapid combination of NO and O2·−(13), inactivates prostacyclin (PGI2) synthase via chemical nitration, leaving PGH2unmetabolized, which then causes vasospasm and platelet aggregation (38). By reducing O2·−, nebivolol may reduce peroxynitrite and, therefore, reduce vasospasm and platelet aggregation. Finally, ROS have been shown to induce activation of nuclear factor-kB (15), a transcription factor involved in the first steps of atherosclerotic plaque formation (39)and to play a pivotal role in regulating apoptosis (40). Nebivolol, therefore, besides reducing blood pressure, may help prevent atherosclerotic initiation and complications.
☆ Supported, in part, by grants from Menarini Phamaceutical; the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation.
- bovine aortic endothelial cells
- BLOX-1-CHO cells
- CHO-K1 cells stably expressing bovine LOX-1
- bLOX-1 mAb
- anti-bovine LOX-1 monoclonal antibody
- coronary artery disease
- CHO-K1 cells
- Chinese hamster ovary-K1 cells
- endothelial nitric oxide synthase
- hLOX-1 mAb
- anti-human LOX-1 monoclonal antibody
- human umbilical vein endothelial cells
- low-density lipoprotein
- lectin-like ox-LDL receptor-1
- mitogen-activated protein kinase
- nitric oxide
- protein kinase C
- reactive oxygen species
- Received December 31, 2002.
- Revision received June 6, 2003.
- Accepted June 10, 2003.
- American College of Cardiology Foundation
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