Endothelial ;3-Adrenoreceptors Mediate Nitric Oxide–Dependent Vasorelaxation of Coronary Microvessels in Response to the Third-Generation ;-
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《循环学杂志》
the Unit of Pharmacology and Therapeutics (C.D., J.S., P.G., G.D., I.I.L., F.F., C.B., K.J., O.F., J.-L.B.) and the Department of Cardiac Surgery (P.N.), Université Catholique de Louvain Medical School, Brussels, Belgium.
Abstract
Background— The therapeutic effects of nonspecific ;-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective ;1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial ;3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these ;3-adrenoreceptors.
Methods and Results— Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 μmol/L, –86±6% of prostaglandin F2 contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the ;1-2-blocker nadolol, and prevented by the ;1-2-3-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from ;3-adrenoreceptor–deficient mice. Nebivolol (10 μmol/L) also relaxed human coronary microvessels (–71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to ;1-2-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not ;3-adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 μmol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine495, and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by ;1-2-blockade but abrogated by ;1-2-3-blockade (all P<0.05).
Conclusions— Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial ;3-adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.
Key Words: endothelium-derived factors ; microcirculation ; nitric oxide synthase ; receptors, adrenergic, beta ; angiogenesis
Introduction
Nebivolol combines a potent ;1-adrenoreceptor–blocking activity (mostly ascribed to its d-enantiomer) with additional vasodilating properties (attributed to the 2 enantiomers,1 d- and l-nebivolol), thereby providing cardioprotection, as recently demonstrated in the SENIORS trial in heart failure,2 while minimizing peripheral vasoconstriction. Early experimental evidence indicates that the drug’s vasodilating effects depend on endothelium-dependent mechanisms3 as inferred from their attenuation by nitric oxide synthase (NOS) and soluble guanylyl cyclase inhibitors3–6 in different vascular beds and species, including humans.7–9 However, the nature of the transduction pathway as well as the identity of the potential receptor that mediates nebivolol’s activation of endothelial nitric oxide synthase (eNOS) leading to vasodilation remain elusive. Neither nebivolol’s ;1-adrenoreceptor nor its -adrenoreceptor or histamine receptor antagonism3 seems to account for its vasodilating properties. Although specific binding of nebivolol to 5-hydroxytryptamine receptors could be measured, methysergide (a 5-hydroxytryptamine1-2 blocker) did not antagonize its vasodilation in dog coronary arteries.3 The stimulation of either ;210- or ;311-adrenoreceptors is known to evoke vasodilation that is, in part, mediated by NO, but the implication for nebivolol’s vasorelaxation remains controversial; ie, nebivolol seems to be devoid of ;2-adrenoreceptor agonistic activity,3,12 although some of its metabolites were suggested to stimulate NO production in mouse aortas through ;2-adrenoreceptor activation.13
Recently, we identified ;3-adrenoreceptors in the endothelium of human coronary resistance microarteries, where they mediate an endothelium-dependent relaxation to both endogenous catecholamines and ;3-adrenoreceptor–preferential agonists.14 We showed this response to be mediated through the production of both NO and a hyperpolarizing factor that partly maintains vessel relaxation when eNOS is inhibited. Such a dual mechanism would be particularly suitable in circumstances of reduced NOS activity or NO bioavailability, as commonly found in atherosclerotic and ischemic diseases. Aside from the regulation of vessel tone, eNOS (and hyperpolarizing factor[s]) modulate several other aspects of vascular biology, including angiogenesis, eg, in response to vascular endothelial growth factor (VEGF). In the context of ischemic cardiac diseases, pharmacological stimulation of NO-dependent neoangiogenesis would offer the additional benefit of increasing capillary density and oxygen supply. Indeed, stimulation of ;3-adrenoreceptors has been shown to increase angiogenesis in other vascular beds, such as the retina.15
We therefore examined whether nebivolol could activate ;3-adrenoreceptors to mediate NO-dependent angiogenic and vasodilatory effects in isolated human and rodent coronary microvessels and characterized the transduction pathway leading to eNOS activation.
Methods
Animals
Experiments were conducted in male Wistar rats (2 to 3 months-old) or in 8-week-old male, homozygous mice deficient for either eNOS (NOS3, B6.129P2tm1Unc; Jackson Laboratories, Bar Harbor, Me) or the ;3-adrenoreceptor (Ardb3tm1Lowl,16 kindly provided by Drs P. Valet [INSERM, Hopital Rangueil, Toulouse, France) and B.B. Lowell (Harvard Medical School, Boston, Mass]). Age-matched, wild-type (C57BL/6J background for the NOS3-deficient mice and FVB/n background for the ;3-deficient mice) littermates were used as controls. Animals were housed in a temperature-controlled animal facility with a 12-hour light/dark cycle, with tap water and rodent chow provided ad libitum. All experimental protocols were approved by the local Ethics Committee.
Videomotion Analysis of Vessel Contraction
Coronary microarteries were carefully dissected from freshly isolated rat or mouse hearts or from human right atrial appendages obtained during cardiac surgery. Samples were obtained from patients (n=17) undergoing either cardiac surgery for coronary artery bypass graft and valve replacement or repair (or a combination thereof for some patients). All tissue collection procedures were approved by the local Ethics Committee. The external diameter of pressurized microvessels was continuously monitored as previously described.14 All experiments were performed in the presence of indomethacin. After equilibration (45 to 60 minutes), vessels were challenged with a high-KCl solution to assess their viability, and the presence of a functional endothelium was determined from their relaxation to substance P.
BAEC Culture
Bovine aortic endothelial cells (BAECs) purchased from Clonetics were grown according to standard procedures and used between passages 4 and 13. Serum-starved BAECs were exposed to nebivolol (0.1 to 10 μmol/L) or its solvent after a 30- to 60-minute preincubation with various pharmacological modulators, ie, 1 to 5 mmol/L NG-nitro-L-arginine methyl ester (L-NAME), bupranolol (1 to 10 μmol/L), or nadolol (1 to 10 μmol/L).
Measurement of NO Production by Amperometric Electrode
NO production was first evaluated by the quantity of nitrite/nitrate (NOx) detected in the cell-culture supernatant as follows. After reduction of nitrates with a chemical nitrate reducer (Nitralyser, World Precision Instruments), the nitrites were chemically reduced to NO, the concentration of which was determined with an NO-specific polarographic probe (ISO-NO from World Precision Instruments)17 and normalized to the protein content of each cell.
Measurement of NO Produced in ECs by EPR Spectroscopy
NO produced by BAECs was assessed by electron paramagnetic resonance (EPR) spin trapping as follows. NO was trapped by a colloid of diethyldithiocarbamate complexed with iron [Fe(II)(DETC)2], and the EPR signal of the formed paramagnetic complex [Fe(II)NO(DETC)2] was quantified as reported previously.18,19 EPR measurements were performed on a Bruker EMX EPR spectrometer (X-band) under the following conditions: temperature 77K, microwave frequency 9.35 GHz, microwave power 20 mW, modulation frequency 100 kHz, and modulation amplitude 0.5 mT. The third component of the EPR signal was used for relative comparison of the concentrations of trapped NO in the samples.
Measurement of [Ca2+]i in ECs
[Ca2+]i was determined with the fluorescent calcium indicator fura-2 as described previously.17 Fura-2–loaded BAECs were kept in an organ chamber at 37°C and placed on the stage of a Zeiss Axiovert-100 microscope connected to a xenon lamp that allows excitation of the sample with wavelengths alternating between 340 and 380 nm (frequency, 2.2 Hz). The emitted fluorescence signal from fura-2 was collected through a 500-nm filter (Ionoptix System). Results were expressed as increases in the 340-380 fluorescence ratios.
Immunodetection of Phosphorylated Protein eNOS
Nebivolol-, bradykinin-, or VEGF-treated (VEGF from R&D Systems) cell cultures were rinsed with Hank’s balanced salt solution, scraped, and lysed by sonication in lysis buffer containing phosphatase and protease inhibitors (composition in mmol/L: imidazole, 50, pH 7.0; KCl, 300; NaF, 10; EGTA, 1; MgCl2, 0.5; ;-glycerophosphate, 10; NaVO4, 1; dithiothreitol, 1; phenylmethylsulfonyl fluoride, 0.1; and benzamidine, 1). Equal amounts of protein were separated by 8% acrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and then blocked overnight in 0.1% Tris-buffered saline/Tween-20 buffer containing 5% (wt/vol) nonfat dry milk. Signals for phosphorylated eNOS and eNOS (measured on the same membranes) were obtained by immunodetection as previously described.17 Anti-phosphothreonine 495 (Thr495), anti-phosphoserine 1177 (Ser1177), and anti-eNOS monoclonal antibodies were purchased from Upstate Cell Signaling, Cell Signaling Technologies, and BD Transduction Laboratories, respectively. Results were expressed as phosphorylated eNOS to eNOS ratios.
Preparation of 3D Aortic Ring Cultures in a Collagen Substrate
Blood was collected with a 26-gauge needle from the right ventricle of anesthetized mice and was centrifuged (5 minutes, 3000 rpm) to obtain autologous serum. The thoracic aortas were collected and transferred to ice-cold, serum-free Dulbecco’s minimum essential medium (GIBCO). Cleaned aortic rings (1 mm long) were embedded in rat tail interstitial collagen gel (1.5 mg/mL), prepared by mixing 7.5 volumes of 2 mg/mL collagen (Collagen R, Serva), 1 volume of 10x minimal essential medium (Promo Cells), and 1.5 volumes of NaHCO3 (15.6 mg/mL), with pH adjusted to 7.4. Collagen gels containing the aortic rings were polymerized in cylindrical agarose wells and kept in triplicate at 37°C in Petri dishes filled with 6 mL of MCDB131 (Life Technologies Ltd) supplemented with 25 mmol/L NaHCO3, 2.5% autologous serum, 1% glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Digital images of microvessel outgrowths were obtained with use of a Zeiss microscope (Axiovert 25) coupled to a video camera (Pixera Pro) on days 5, 7, 9, and 12.
Drugs
Stock solutions of nebivolol were prepared in dimethyl sulfoxide (DMSO) at concentrations of 10 to 30 mmol/L. Subsequent dilutions were prepared in physiological solution (composition as detailed earlier). Identical dilutions of the solvent (DMSO) were always prepared in parallel and used as controls for the effect of the active drug.
Statistics
All results are expressed as mean±SEM. Statistical comparisons were analyzed by Student’s t test or ANOVA, followed by a Bonferroni or Dunnett post test where appropriate. A value of P<0.05 was considered significant.
Results
Nebivolol-Induced Relaxation of Human Coronary Microarteries
Human coronary microarteries precontracted with 10 nmol/L endothelin-1 were subjected to cumulative additions of nebivolol. Nebivolol relaxed most of the agonist-evoked contraction (n=3; residual contraction, 28.5±5.4%; Figure 1A). Similar microarteries were further precontracted with a high-KCl solution to exclude possible interactions with any endothelium-derived hyperpolarizing factor–mediated relaxation. As illustrated in Figure 1B and 1C, 10 μmol/L nebivolol still relaxed 79±9% of the maximum contraction evoked by KCl (n=6). This relaxation was sensitive to NOS inhibition with L-nitroarginine (100 μmol/L; to 31.2±9.3% relaxation; n=6, P<0.05). Vessels that lacked endothelium-dependent relaxation to substance P failed to relax with nebivolol (Figure 1B, upper right; single measurement at 10 μmol/L; n=3). Blockade of specific ;-adrenoreceptors was then tested on the relaxation to nebivolol. As shown in Figure 1D, pretreatment of human coronary microarteries with the ;1-2-blocker nadolol (10 μmol/L) had no effect on nebivolol-induced vasodilation. This clearly identified an endothelium- and NO-dependent vasodilation that did not involve activation of ;1-2-adrenoreceptors.
Nebivolol-Induced Relaxation: Involvement of a ;3-Adrenergic Pathway
The insensitivity to nadolol suggested involvement of another ;-adrenoreceptor isotype. Because we had previously demonstrated expression of ;3-adrenoreceptors in rodent and human coronary microarteries, involvement of this receptor was further studied with pharmacological and genetic approaches in rodent vessels. Relaxation to nebivolol (0.3 to 10 μmol/L) was first examined in rat coronary microarteries, in which the maximum relaxation amounted to 86±6% (n=7) of the maximal contraction to prostaglandin F2 (Figure 2A). This relaxation was also partly sensitive to NOS inhibition (not shown).
As in human vessels, preincubation with the ;1-2-specific antagonist nadolol did not significantly modify nebivolol-induced vasodilation (relaxation, 77±8%; n=3, P>0.05), whereas the complete ;1-2-3-blocker bupranolol significantly inhibited nebivolol’s effect (residual relaxation, 21±5%; n=3, P<0.01).
To further confirm a ;3-adrenergic effect, relaxations to nebivolol were compared in coronary microarteries from mice genetically deficient in ;3-adrenoceptors (;3-AR–/–) and their wild-type controls. Although nebivolol almost fully relaxed vessels from the controls (relaxation, 90.8±2.9%; n=5), its effect was greatly reduced in coronary microarteries from ;3-AR–/– mice (relaxation, 31.1±8.6%; n=6, P<0.001).
Nebivolol Induced an Angiogenic Response Through NO and ;3-Adrenoreceptor Stimulation
The relaxation assays suggested that nebivolol activates endothelial NO production through ;3-adrenoreceptor stimulation. Next, we tested whether the same effects would promote a NO-dependent angiogenic response in mouse aortic rings ex vivo. Initial experiments in rings from C57BL/6J mice indicated that 3 μmol/L nebivolol increased the number of microtubes growing out from the rings (day 12, 18.1±2.3, n=16) whereas corresponding amounts of the solvent (0.03% DMSO) had no effect (9.1±1.05; n=15, P<0.001).
To assess the involvement of ;3-adrenoreceptors in the angiogenic response, we tested the effects of nebivolol (3 μmol/L) and VEGF (40 ng/mL) on aortic rings from ;3-AR–/– mice and their controls. As shown in Figure 3B, in control mice, VEGF stimulated microtube formation compared with medium alone (20.5±2.6 versus 8.0±1.1 at day 12; n=11), as did 3 μmol/L nebivolol (day 12, 16.7±1.9, n=10 versus 6.1±1.2, n=8). By contrast, in rings from ;3-AR–/– mice, the effect of nebivolol was lost (8.0±1.1 versus 8.0±0.6 with DMSO; n=10), whereas VEGF still induced microtube outgrowths (18.2±3.3 versus 7.7±1.1 with solvent; n=11, P<0.001; Figure 3D).
To assess the involvement of NO in the angiogenic response, we tested the effects of VEGF and nebivolol on rings from eNOS-deficient mice. In these animals, the spontaneous microtube growth was not altered, but both VEGF and nebivolol failed to increase their number (Figure 3C), whereas both drugs did so in rings from control C57BL/6J animals (Figure 3A). This clearly implicated NO in the effects mediated by nebivolol on angiogenesis.
Nebivolol-Evoked Calcium Increases and NOx Production in ECs
To directly confirm that nebivolol stimulates endothelial NO production, we used cultured BAECs, in which we previously verified the expression of both ;3-adrenoreceptors and eNOS. As shown in Figure 4B, 1 μmol/L nebivolol increased the formation of NO by as much as 244±32% (n=3) of basal level, as assessed by EPR spin trapping. Incubation of the cells with the NOS inhibitor L-NAME (1 mmol/L) decreased this NO production to 61±31% (n=3) of controls. The permeable superoxide dismutase mimetic MnTBAP (50 μmol/L) did not significantly alter NO production in this setting (n=3), indicating that nebivolol increased eNOS activity without modulating superoxide anion production. These data were confirmed with a second method of measurement of NO production with a polarographic probe (Figure 4A).
Stimulation of BAECs with nebivolol also evoked a sustained (and dose-dependent; not shown) increase in calcium signal (Figure 4C), with an amplitude similar to that evoked by bradykinin, a well-known endothelial agonist (not shown). Again, as in the relaxation assays, this calcium signal was insensitive to ;1-2-adrenoreceptor blockade with nadolol (n=5) but was abrogated by ;1-2-3-adrenoreceptor blockade with bupranolol (n=5; Figure 4C).
Effect of Nebivolol on the Phosphorylation of eNOS
Calcium-dependent activation of eNOS is followed by changes in its phosphorylation state on several regulatory residues. Some (but not all) agonists that activate eNOS induce phosphorylation of eNOS on Ser1177. Accordingly, treatment of BAECs with VEGF (40 ng/mL; 5 minutes) induced the phosphorylation of eNOS on Ser1177. However, treatment with 1 μmol/L nebivolol under similar conditions failed to increase the phosphorylation level of Ser1177 (Figure 5A). Other agonists such as bradykinin activate eNOS through transient dephosphorylation of Thr495. In our BAECs, 100 nmol/L bradykinin elicited a transient decrease in phosphorylation of Thr495 to 44.6±4.5% (n=4) of the basal level, with the lowest level within 1 minute and recovery to basal level within 3 minutes (not shown). Similarly, 1 μmol/L nebivolol elicited the dephosphorylation of eNOS on Thr495, with a decrease to 42.6±17.4% (n=4, P<0.05) and 26.5±15.5% (n=3, P<0.05) of baseline after 3 and 10 minutes, respectively. DMSO (0.03%) did not modify Thr495 phosphorylation (Figure 5B).
Discussion
The current study provides several new insights in the understanding of the vascular effects of the ;1-adrenoreceptor antagonist nebivolol and a characterization of the underlying mechanisms: (1) nebivolol exerts a potent endothelium-dependent vasodilation of human and rodent coronary resistance arteries, as well as proangiogenic effects in vitro; (2) these effects are mediated through the stimulation of ;3-adrenoreceptors and NO production; and (3) these effects are accompanied by a rise in cytosolic calcium and an activating dephosphorylation of eNOS on Thr495 in ECs.
Whereas previous studies have shown a vasodilating effect of nebivolol in the human brachial vascular bed in vivo,7–9,20 our results demonstrate for the first time a very potent vasodilating effect of the drug in human coronary microvessels (70 to 170 μm in diameter), a major site of regulation of coronary resistance and perfusion reserve. This is of potential therapeutic importance, given the prognostic value of coronary microvascular dysfunction in human cardiomyopathies.21 As in other vascular beds,3,7,8 this effect is dependent on the presence of a functional endothelium and is sensitive to NOS inhibitors, but beyond previous studies, we now provide direct demonstration of NO release from ECs treated with nebivolol by 2 different techniques (Figure 4A and 4B).
Treatment of rat and human coronary microarteries with nadolol (a ;1-2-adrenoreceptor–specific blocker) did not significantly attenuate the relaxation to nebivolol, confirming its independence from the drug’s binding on ;1- or ;2-adrenoreceptors. Consistent with the results of Trochu and collaborators11 in the rat aorta, our previous study14 demonstrated that in human coronary microarteries, norepinephrine and a ;3-adrenoreceptor–preferential agonist, BRL 37344, evoked a sustained vasodilation that was partly mediated by NO. This was confirmed by the demonstration of specific immunohistochemical staining for ;3-adrenoreceptor proteins on the endothelium of the same vessels. We now show that nebivolol produces its vasodilation in similar preparations through interaction with this ;3-adrenoreceptor isotype. Indeed, contrary to the neutral effect of ;1-2-blockade in human (Figure 1D) and rodent (Figure 2A) vessels, the complete ;1-2-3-blocker bupranolol did significantly inhibit the relaxation to nebivolol in rat coronary microarteries (Figure 2A). Again, these results were fully consistent with the direct endothelial response to nebivolol in vitro (Figure 4C). Finally, the involvement of this ;3-isotype in nebivolol’s effect was definitively demonstrated by the impaired relaxation in aortas from mice genetically deficient in ;3-adrenoreceptors (Figure 2B). The identification of partial agonism on ;3-adrenoreceptors provides new mechanistic insights into the pharmacodynamic effect of this third-generation ;1-adrenoreceptor blocker, with coupling to critical ancillary effects on the vasculature.
NO is recognized as a major downstream effector of proangiogenic cytokines, such as VEGF, in ECs.22 Because we directly observed increased NO production upon stimulation with nebivolol in ECs, we tested whether the drug would also promote their adoption of a proangiogenic phenotype in vitro. We demonstrated that nebivolol induced microtube formation in isolated aortic rings, indicating proangiogenic properties (Figure 3). Our comparative use of rings from ;3-AR–/– and eNOS–/– mice again confirmed that this property depends on the presence of both eNOS and ;3-adrenoreceptors (Figure 3C and 3D). Although another study has shown that ;3-adrenoreceptor stimulation initiated proliferation and migration of ECs, 2 initial steps toward angiogenesis,15 we now directly demonstrate tube outgrowth from full, mature vessels clearly associated with ;3-adrenoreceptor stimulation by nebivolol.
Understanding of the molecular mechanisms of eNOS activation has recently progressed through the identification of regulatory phosphorylation sites that modulate eNOS activity (eg, Ser1177, Thr495) after increases in intracellular calcium. We previously demonstrated sequential events leading to eNOS activation on calcium-dependent dissociation of eNOS from caveolin-1, followed by recruitment of protein kinase Akt/PKB, resulting in phosphorylation of eNOS on Ser1177.17,23 The latter was strictly dependent on a rise in intracellular calcium concentration. Therefore, we examined the ability of nebivolol to interfere with several of these activation events. Our results demonstrate that when added to cultured ECs, nebivolol evoked a strong increase in fura-2 calcium signal (Figure 4C) of an amplitude similar to that of other endothelial agonists, such as bradykinin (not shown). However, we did not detect changes in phosphorylation of eNOS on Ser1177, suggesting that in our cells and experimental conditions, activation of eNOS by nebivolol does not involve phosphorylation of this particular residue. Conversely, we demonstrated that nebivolol induced dephosphorylation of the inhibitory phospho-Thr495 within the calcium-calmodulin consensus binding sequence of eNOS. In ECs activated with Ca2+-elevating agonists such as bradykinin (or nebivolol, as in our study), substantially more activating calmodulin binds to eNOS when Thr495 is dephosphorylated.24 Of note, this dephosphorylation on Thr495 was not accompanied by an increase in production of superoxide anions (as shown by insensitivity of the EPR signal for NO to a superoxide dismutase analogue; Figure 4B), as would have been anticipated from uncoupled eNOS.25
Potential Limitations and Pathophysiological Implications
Our results were obtained with nebivolol at a range of concentrations identical to those previously reported in the literature.3,26 Although it could be argued that concentrations used in vitro are higher than those achieved in patients, the effects of ;3-agonism presented here recapitulate the increases in human brachial blood flow7,9 observed at doses of nebivolol currently used therapeutically, which were also shown to be mediated by increases in NO production. Our data now suggest the possibility of similar effects in the human coronary vasculature. Broeders and collegues13 also demonstrated that sera from mice treated with nebivolol activated NO production in mouse aortic segments, suggesting the involvement of active metabolites, at least in mice. Although their paradigms indicated a ;2-adrenoreceptor effect, this was exclusively inferred from the use of butoxamine in mouse aortas. No other mouse or human vessel bed was examined.
The NO-releasing and vasodilating properties of nebivolol in coronary microvessels may underlie its beneficial effects in patients with ischemic or dilated cardiomyopathies,2 particularly those with diastolic dysfunction,27 given the direct lusitropic properties of NO in the myocardium.28 Diseases such as dyslipidemia, diabetes, and atherosclerosis are all associated with a decrease in eNOS expression, NO bioavailability, or both (due to, eg, oxidative stress), which could limit the clinical impact of a drug that depends on NO release. Importantly, in human coronary arterioles, we demonstrated that in situations where NO production is impaired, endothelium-derived hyperpolarizing factor(s) can fully compensate for the loss of NO and completely restore ;3-adrenoreceptor–mediated relaxation.14 This and the relative resistance of the ;3-adrenoreceptors to homologous desensitization29 would seem of particular importance in preserving myocardial perfusion in circumstances such as ischemia and heart failure. Finally, if reflective of similar properties in vivo, the proangiogenic effect of nebivolol would provide additional cardiovascular benefits in patients with ischemic cardiac or peripheral diseases.
In conclusion, our work demonstrates for the first time that nebivolol is able to elicit a potent and sustained relaxation of human coronary resistance arteries that is at least partially NO mediated. Our results obtained in both ECs and isolated coronary arteries substantiate the hypothesis that nebivolol exerts an agonist activity on ;3-adrenoreceptors to induce sustained NO production through increases in cytosolic calcium concentrations and dephosphorylation of Thr495-eNOS.
Acknowledgments
This work was supported by grants ARC 01/06-271 and PAI-P5/02 to Dr Balligand. Drs Dessy and Feron are research associates of the Fonds National de la Recherche Scientifique (FNRS); Julie Saliez and Fran;oise Frérart are recipients of a FRIA fellowship; Dr Belge and Géraldine Daneau are fellows of the FNRS. Part of this work was supported by Menarini Industrie, Firenze, Italy.
References
Van Lommen G, De Bruyn M, Schroven M. Synthesis and pharmacological properties of nebivolol, a new antihypertensive compound. J Pharm Belg. 1990; 45: 355–360.
Flather MD, Shibata MC, Coats AJ, Van Veldhuisen DJ, Parkhomenko A, Borbola J, Cohen-Solal A, Dumitrascu D, Ferrari R, Lechat P, Soler-Soler J, Tavazzi L, Spinarova L, Toman J, Bohm M, Anker SD, Thompson SG, Poole-Wilson PA. Randomized trial to determine the effect of nebivolol on mortality and cardiovascular hospital admission in elderly patients with heart failure (SENIORS). Eur Heart J. 2005; 26: 215–225.
Gao YS, Nagao T, Bond RA, Janssens WJ, Vanhoutte PM. Nebivolol induces endothelium-dependent relaxations of canine coronary arteries. J Cardiovasc Pharmacol. 1991; 17: 964–969.
Cosentino F, Bonetti S, Rehorik R, Eto M, Werner-Felmayer G, Volpe M, Luscher TF. Nitric-oxide-mediated relaxations in salt-induced hypertension: effect of chronic ;1-selective receptor blockade. J Hypertens. 2002; 20: 421–428.
Altwegg LA, d’Uscio LV, Barandier C, Cosentino F, Yang Z, Luscher TF. Nebivolol induces NO-mediated relaxations of rat small mesenteric but not of large elastic arteries. J Cardiovasc Pharmacol. 2000; 36: 316–320.
Ignarro LJ, Byrns RE, Trinh K, Sisodia M, Buga GM. Nebivolol: a selective ;1-adrenergic receptor antagonist that relaxes vascular smooth muscle by nitric oxide- and cyclic GMP-dependent mechanisms. Nitric Oxide. 2002; 7: 75–82.
Ritter JM. Nebivolol: endothelium-mediated vasodilating effect. J Cardiovasc Pharmacol. 2001; 38 (suppl 3): S13–S16.
Cockcroft JR, Chowienczyk PJ, Brett SE, Chen CP, Dupont AG, Van Nueten L, Wooding SJ, Ritter JM. Nebivolol vasodilates human forearm vasculature: evidence for an L-arginine/NO-dependent mechanism. J Pharmacol Exp Ther. 1995; 274: 1067–1071.
Dawes M, Brett SE, Chowienczyk PJ, Mant TG, Ritter JM. The vasodilator action of nebivolol in forearm vasculature of subjects with essential hypertension. Br J Clin Pharmacol. 1999; 48: 460–463.
Gray DW, Marshall I. Novel signal transduction pathway mediating endothelium-dependent ;-adrenoceptor vasorelaxation in rat thoracic aorta. Br J Pharmacol. 1992; 107: 684–690.
Trochu JN, Leblais V, Rautureau Y, Beverelli F, Le Marec H, Berdeaux A, Gauthier C. ;3-Adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium-derived nitric oxide in rat thoracic aorta. Br J Pharmacol. 1999; 128: 69–76.
Gosgnach W, Boixel C, Nevo N, Poiraud T, Michel JB. Nebivolol induces calcium-independent signaling in endothelial cells by a possible ;-adrenergic pathway. J Cardiovasc Pharmacol. 2001; 38: 191–199.
Broeders MA, Doevendans PA, Bekkers BC, Bronsaer R, van Gorsel E, Heemskerk JW, Egbrink MG, van Breda E, Reneman RS, van Der Zee R. Nebivolol: a third-generation ;-blocker that augments vascular nitric oxide release: endothelial ;2-adrenergic receptor-mediated nitric oxide production. Circulation. 2000; 102: 677–684.
Dessy C, Moniotte S, Ghisdal P, Havaux X, Noirhomme P, Balligand JL. Endothelial ;3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization. Circulation. 2004; 110: 948–954.
Steinle JJ, Booz GW, Meininger CJ, Day JN, Granger HJ. ;3-adrenergic receptors regulate retinal endothelial cell migration and proliferation. J Biol Chem. 2003; 278: 20681–20686.
Susulic VS, Frederich RC, Lawitts J, Tozzo E, Kahn BB, Harper ME, Himms-Hagen J, Flier JS, Lowell BB. Targeted disruption of the ;3-adrenergic receptor gene. J Biol Chem. 1995; 270: 29483–29492.
Brouet A, Sonveaux P, Dessy C, Balligand JL, Feron O. Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells. J Biol Chem. 2001; 276: 32663–32669.
Vanin AF. Iron diethyldithiocarbamate as spin trap for nitric oxide detection. Methods Enzymol. 1999; 301: 269–279.
Kleschyov AL, Munzel T. Advanced spin trapping of vascular nitric oxide using colloid iron diethyldithiocarbamate. Methods Enzymol. 2002; 359: 42–51.
Kubli S, Feihl F, Waeber B. ;-Blockade with nebivolol enhances the acetylcholine-induced cutaneous vasodilation. Clin Pharmacol Ther. 2001; 69: 238–244.
Cecchi F, Olivotto I, Gistri R, Lorenzoni R, Chiriatti G, Camici PG. Coronary microvascular dysfunction and prognosis in hypertrophic cardiomyopathy. N Engl J Med. 2003; 349: 1027–1035.
Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997; 100: 3131–3139.
Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res. 2001; 89: 866–873.
Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: e68–e75.
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
Parenti A, Filippi S, Amerini S, Granger HJ, Fazzini A, Ledda F. Inositol phosphate metabolism and nitric-oxide synthase activity in endothelial cells are involved in the vasorelaxant activity of nebivolol. J Pharmacol Exp Ther. 2000; 292: 698–703.
De Cree J, Vanwelden H, Geukens H, Verhaegen H. The effect of nebivolol in patients with left ventricular diastolic dysfunction. Drug Invest. 1991; 3: 142–143.
Massion PB, Balligand JL. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol. 2003; 546: 63–75.
Liggett SB, Freedman NJ, Schwinn DA, Lefkowitz RJ. Structural basis for receptor subtype-specific regulation revealed by a chimeric ;3/;2-adrenergic receptor. Proc Natl Acad Sci U S A. 1993; 90: 3665–3669.(Chantal Dessy, PhD; Julie)
Abstract
Background— The therapeutic effects of nonspecific ;-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective ;1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial ;3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these ;3-adrenoreceptors.
Methods and Results— Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 μmol/L, –86±6% of prostaglandin F2 contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the ;1-2-blocker nadolol, and prevented by the ;1-2-3-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from ;3-adrenoreceptor–deficient mice. Nebivolol (10 μmol/L) also relaxed human coronary microvessels (–71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to ;1-2-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not ;3-adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 μmol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine495, and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by ;1-2-blockade but abrogated by ;1-2-3-blockade (all P<0.05).
Conclusions— Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial ;3-adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.
Key Words: endothelium-derived factors ; microcirculation ; nitric oxide synthase ; receptors, adrenergic, beta ; angiogenesis
Introduction
Nebivolol combines a potent ;1-adrenoreceptor–blocking activity (mostly ascribed to its d-enantiomer) with additional vasodilating properties (attributed to the 2 enantiomers,1 d- and l-nebivolol), thereby providing cardioprotection, as recently demonstrated in the SENIORS trial in heart failure,2 while minimizing peripheral vasoconstriction. Early experimental evidence indicates that the drug’s vasodilating effects depend on endothelium-dependent mechanisms3 as inferred from their attenuation by nitric oxide synthase (NOS) and soluble guanylyl cyclase inhibitors3–6 in different vascular beds and species, including humans.7–9 However, the nature of the transduction pathway as well as the identity of the potential receptor that mediates nebivolol’s activation of endothelial nitric oxide synthase (eNOS) leading to vasodilation remain elusive. Neither nebivolol’s ;1-adrenoreceptor nor its -adrenoreceptor or histamine receptor antagonism3 seems to account for its vasodilating properties. Although specific binding of nebivolol to 5-hydroxytryptamine receptors could be measured, methysergide (a 5-hydroxytryptamine1-2 blocker) did not antagonize its vasodilation in dog coronary arteries.3 The stimulation of either ;210- or ;311-adrenoreceptors is known to evoke vasodilation that is, in part, mediated by NO, but the implication for nebivolol’s vasorelaxation remains controversial; ie, nebivolol seems to be devoid of ;2-adrenoreceptor agonistic activity,3,12 although some of its metabolites were suggested to stimulate NO production in mouse aortas through ;2-adrenoreceptor activation.13
Recently, we identified ;3-adrenoreceptors in the endothelium of human coronary resistance microarteries, where they mediate an endothelium-dependent relaxation to both endogenous catecholamines and ;3-adrenoreceptor–preferential agonists.14 We showed this response to be mediated through the production of both NO and a hyperpolarizing factor that partly maintains vessel relaxation when eNOS is inhibited. Such a dual mechanism would be particularly suitable in circumstances of reduced NOS activity or NO bioavailability, as commonly found in atherosclerotic and ischemic diseases. Aside from the regulation of vessel tone, eNOS (and hyperpolarizing factor[s]) modulate several other aspects of vascular biology, including angiogenesis, eg, in response to vascular endothelial growth factor (VEGF). In the context of ischemic cardiac diseases, pharmacological stimulation of NO-dependent neoangiogenesis would offer the additional benefit of increasing capillary density and oxygen supply. Indeed, stimulation of ;3-adrenoreceptors has been shown to increase angiogenesis in other vascular beds, such as the retina.15
We therefore examined whether nebivolol could activate ;3-adrenoreceptors to mediate NO-dependent angiogenic and vasodilatory effects in isolated human and rodent coronary microvessels and characterized the transduction pathway leading to eNOS activation.
Methods
Animals
Experiments were conducted in male Wistar rats (2 to 3 months-old) or in 8-week-old male, homozygous mice deficient for either eNOS (NOS3, B6.129P2tm1Unc; Jackson Laboratories, Bar Harbor, Me) or the ;3-adrenoreceptor (Ardb3tm1Lowl,16 kindly provided by Drs P. Valet [INSERM, Hopital Rangueil, Toulouse, France) and B.B. Lowell (Harvard Medical School, Boston, Mass]). Age-matched, wild-type (C57BL/6J background for the NOS3-deficient mice and FVB/n background for the ;3-deficient mice) littermates were used as controls. Animals were housed in a temperature-controlled animal facility with a 12-hour light/dark cycle, with tap water and rodent chow provided ad libitum. All experimental protocols were approved by the local Ethics Committee.
Videomotion Analysis of Vessel Contraction
Coronary microarteries were carefully dissected from freshly isolated rat or mouse hearts or from human right atrial appendages obtained during cardiac surgery. Samples were obtained from patients (n=17) undergoing either cardiac surgery for coronary artery bypass graft and valve replacement or repair (or a combination thereof for some patients). All tissue collection procedures were approved by the local Ethics Committee. The external diameter of pressurized microvessels was continuously monitored as previously described.14 All experiments were performed in the presence of indomethacin. After equilibration (45 to 60 minutes), vessels were challenged with a high-KCl solution to assess their viability, and the presence of a functional endothelium was determined from their relaxation to substance P.
BAEC Culture
Bovine aortic endothelial cells (BAECs) purchased from Clonetics were grown according to standard procedures and used between passages 4 and 13. Serum-starved BAECs were exposed to nebivolol (0.1 to 10 μmol/L) or its solvent after a 30- to 60-minute preincubation with various pharmacological modulators, ie, 1 to 5 mmol/L NG-nitro-L-arginine methyl ester (L-NAME), bupranolol (1 to 10 μmol/L), or nadolol (1 to 10 μmol/L).
Measurement of NO Production by Amperometric Electrode
NO production was first evaluated by the quantity of nitrite/nitrate (NOx) detected in the cell-culture supernatant as follows. After reduction of nitrates with a chemical nitrate reducer (Nitralyser, World Precision Instruments), the nitrites were chemically reduced to NO, the concentration of which was determined with an NO-specific polarographic probe (ISO-NO from World Precision Instruments)17 and normalized to the protein content of each cell.
Measurement of NO Produced in ECs by EPR Spectroscopy
NO produced by BAECs was assessed by electron paramagnetic resonance (EPR) spin trapping as follows. NO was trapped by a colloid of diethyldithiocarbamate complexed with iron [Fe(II)(DETC)2], and the EPR signal of the formed paramagnetic complex [Fe(II)NO(DETC)2] was quantified as reported previously.18,19 EPR measurements were performed on a Bruker EMX EPR spectrometer (X-band) under the following conditions: temperature 77K, microwave frequency 9.35 GHz, microwave power 20 mW, modulation frequency 100 kHz, and modulation amplitude 0.5 mT. The third component of the EPR signal was used for relative comparison of the concentrations of trapped NO in the samples.
Measurement of [Ca2+]i in ECs
[Ca2+]i was determined with the fluorescent calcium indicator fura-2 as described previously.17 Fura-2–loaded BAECs were kept in an organ chamber at 37°C and placed on the stage of a Zeiss Axiovert-100 microscope connected to a xenon lamp that allows excitation of the sample with wavelengths alternating between 340 and 380 nm (frequency, 2.2 Hz). The emitted fluorescence signal from fura-2 was collected through a 500-nm filter (Ionoptix System). Results were expressed as increases in the 340-380 fluorescence ratios.
Immunodetection of Phosphorylated Protein eNOS
Nebivolol-, bradykinin-, or VEGF-treated (VEGF from R&D Systems) cell cultures were rinsed with Hank’s balanced salt solution, scraped, and lysed by sonication in lysis buffer containing phosphatase and protease inhibitors (composition in mmol/L: imidazole, 50, pH 7.0; KCl, 300; NaF, 10; EGTA, 1; MgCl2, 0.5; ;-glycerophosphate, 10; NaVO4, 1; dithiothreitol, 1; phenylmethylsulfonyl fluoride, 0.1; and benzamidine, 1). Equal amounts of protein were separated by 8% acrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and then blocked overnight in 0.1% Tris-buffered saline/Tween-20 buffer containing 5% (wt/vol) nonfat dry milk. Signals for phosphorylated eNOS and eNOS (measured on the same membranes) were obtained by immunodetection as previously described.17 Anti-phosphothreonine 495 (Thr495), anti-phosphoserine 1177 (Ser1177), and anti-eNOS monoclonal antibodies were purchased from Upstate Cell Signaling, Cell Signaling Technologies, and BD Transduction Laboratories, respectively. Results were expressed as phosphorylated eNOS to eNOS ratios.
Preparation of 3D Aortic Ring Cultures in a Collagen Substrate
Blood was collected with a 26-gauge needle from the right ventricle of anesthetized mice and was centrifuged (5 minutes, 3000 rpm) to obtain autologous serum. The thoracic aortas were collected and transferred to ice-cold, serum-free Dulbecco’s minimum essential medium (GIBCO). Cleaned aortic rings (1 mm long) were embedded in rat tail interstitial collagen gel (1.5 mg/mL), prepared by mixing 7.5 volumes of 2 mg/mL collagen (Collagen R, Serva), 1 volume of 10x minimal essential medium (Promo Cells), and 1.5 volumes of NaHCO3 (15.6 mg/mL), with pH adjusted to 7.4. Collagen gels containing the aortic rings were polymerized in cylindrical agarose wells and kept in triplicate at 37°C in Petri dishes filled with 6 mL of MCDB131 (Life Technologies Ltd) supplemented with 25 mmol/L NaHCO3, 2.5% autologous serum, 1% glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Digital images of microvessel outgrowths were obtained with use of a Zeiss microscope (Axiovert 25) coupled to a video camera (Pixera Pro) on days 5, 7, 9, and 12.
Drugs
Stock solutions of nebivolol were prepared in dimethyl sulfoxide (DMSO) at concentrations of 10 to 30 mmol/L. Subsequent dilutions were prepared in physiological solution (composition as detailed earlier). Identical dilutions of the solvent (DMSO) were always prepared in parallel and used as controls for the effect of the active drug.
Statistics
All results are expressed as mean±SEM. Statistical comparisons were analyzed by Student’s t test or ANOVA, followed by a Bonferroni or Dunnett post test where appropriate. A value of P<0.05 was considered significant.
Results
Nebivolol-Induced Relaxation of Human Coronary Microarteries
Human coronary microarteries precontracted with 10 nmol/L endothelin-1 were subjected to cumulative additions of nebivolol. Nebivolol relaxed most of the agonist-evoked contraction (n=3; residual contraction, 28.5±5.4%; Figure 1A). Similar microarteries were further precontracted with a high-KCl solution to exclude possible interactions with any endothelium-derived hyperpolarizing factor–mediated relaxation. As illustrated in Figure 1B and 1C, 10 μmol/L nebivolol still relaxed 79±9% of the maximum contraction evoked by KCl (n=6). This relaxation was sensitive to NOS inhibition with L-nitroarginine (100 μmol/L; to 31.2±9.3% relaxation; n=6, P<0.05). Vessels that lacked endothelium-dependent relaxation to substance P failed to relax with nebivolol (Figure 1B, upper right; single measurement at 10 μmol/L; n=3). Blockade of specific ;-adrenoreceptors was then tested on the relaxation to nebivolol. As shown in Figure 1D, pretreatment of human coronary microarteries with the ;1-2-blocker nadolol (10 μmol/L) had no effect on nebivolol-induced vasodilation. This clearly identified an endothelium- and NO-dependent vasodilation that did not involve activation of ;1-2-adrenoreceptors.
Nebivolol-Induced Relaxation: Involvement of a ;3-Adrenergic Pathway
The insensitivity to nadolol suggested involvement of another ;-adrenoreceptor isotype. Because we had previously demonstrated expression of ;3-adrenoreceptors in rodent and human coronary microarteries, involvement of this receptor was further studied with pharmacological and genetic approaches in rodent vessels. Relaxation to nebivolol (0.3 to 10 μmol/L) was first examined in rat coronary microarteries, in which the maximum relaxation amounted to 86±6% (n=7) of the maximal contraction to prostaglandin F2 (Figure 2A). This relaxation was also partly sensitive to NOS inhibition (not shown).
As in human vessels, preincubation with the ;1-2-specific antagonist nadolol did not significantly modify nebivolol-induced vasodilation (relaxation, 77±8%; n=3, P>0.05), whereas the complete ;1-2-3-blocker bupranolol significantly inhibited nebivolol’s effect (residual relaxation, 21±5%; n=3, P<0.01).
To further confirm a ;3-adrenergic effect, relaxations to nebivolol were compared in coronary microarteries from mice genetically deficient in ;3-adrenoceptors (;3-AR–/–) and their wild-type controls. Although nebivolol almost fully relaxed vessels from the controls (relaxation, 90.8±2.9%; n=5), its effect was greatly reduced in coronary microarteries from ;3-AR–/– mice (relaxation, 31.1±8.6%; n=6, P<0.001).
Nebivolol Induced an Angiogenic Response Through NO and ;3-Adrenoreceptor Stimulation
The relaxation assays suggested that nebivolol activates endothelial NO production through ;3-adrenoreceptor stimulation. Next, we tested whether the same effects would promote a NO-dependent angiogenic response in mouse aortic rings ex vivo. Initial experiments in rings from C57BL/6J mice indicated that 3 μmol/L nebivolol increased the number of microtubes growing out from the rings (day 12, 18.1±2.3, n=16) whereas corresponding amounts of the solvent (0.03% DMSO) had no effect (9.1±1.05; n=15, P<0.001).
To assess the involvement of ;3-adrenoreceptors in the angiogenic response, we tested the effects of nebivolol (3 μmol/L) and VEGF (40 ng/mL) on aortic rings from ;3-AR–/– mice and their controls. As shown in Figure 3B, in control mice, VEGF stimulated microtube formation compared with medium alone (20.5±2.6 versus 8.0±1.1 at day 12; n=11), as did 3 μmol/L nebivolol (day 12, 16.7±1.9, n=10 versus 6.1±1.2, n=8). By contrast, in rings from ;3-AR–/– mice, the effect of nebivolol was lost (8.0±1.1 versus 8.0±0.6 with DMSO; n=10), whereas VEGF still induced microtube outgrowths (18.2±3.3 versus 7.7±1.1 with solvent; n=11, P<0.001; Figure 3D).
To assess the involvement of NO in the angiogenic response, we tested the effects of VEGF and nebivolol on rings from eNOS-deficient mice. In these animals, the spontaneous microtube growth was not altered, but both VEGF and nebivolol failed to increase their number (Figure 3C), whereas both drugs did so in rings from control C57BL/6J animals (Figure 3A). This clearly implicated NO in the effects mediated by nebivolol on angiogenesis.
Nebivolol-Evoked Calcium Increases and NOx Production in ECs
To directly confirm that nebivolol stimulates endothelial NO production, we used cultured BAECs, in which we previously verified the expression of both ;3-adrenoreceptors and eNOS. As shown in Figure 4B, 1 μmol/L nebivolol increased the formation of NO by as much as 244±32% (n=3) of basal level, as assessed by EPR spin trapping. Incubation of the cells with the NOS inhibitor L-NAME (1 mmol/L) decreased this NO production to 61±31% (n=3) of controls. The permeable superoxide dismutase mimetic MnTBAP (50 μmol/L) did not significantly alter NO production in this setting (n=3), indicating that nebivolol increased eNOS activity without modulating superoxide anion production. These data were confirmed with a second method of measurement of NO production with a polarographic probe (Figure 4A).
Stimulation of BAECs with nebivolol also evoked a sustained (and dose-dependent; not shown) increase in calcium signal (Figure 4C), with an amplitude similar to that evoked by bradykinin, a well-known endothelial agonist (not shown). Again, as in the relaxation assays, this calcium signal was insensitive to ;1-2-adrenoreceptor blockade with nadolol (n=5) but was abrogated by ;1-2-3-adrenoreceptor blockade with bupranolol (n=5; Figure 4C).
Effect of Nebivolol on the Phosphorylation of eNOS
Calcium-dependent activation of eNOS is followed by changes in its phosphorylation state on several regulatory residues. Some (but not all) agonists that activate eNOS induce phosphorylation of eNOS on Ser1177. Accordingly, treatment of BAECs with VEGF (40 ng/mL; 5 minutes) induced the phosphorylation of eNOS on Ser1177. However, treatment with 1 μmol/L nebivolol under similar conditions failed to increase the phosphorylation level of Ser1177 (Figure 5A). Other agonists such as bradykinin activate eNOS through transient dephosphorylation of Thr495. In our BAECs, 100 nmol/L bradykinin elicited a transient decrease in phosphorylation of Thr495 to 44.6±4.5% (n=4) of the basal level, with the lowest level within 1 minute and recovery to basal level within 3 minutes (not shown). Similarly, 1 μmol/L nebivolol elicited the dephosphorylation of eNOS on Thr495, with a decrease to 42.6±17.4% (n=4, P<0.05) and 26.5±15.5% (n=3, P<0.05) of baseline after 3 and 10 minutes, respectively. DMSO (0.03%) did not modify Thr495 phosphorylation (Figure 5B).
Discussion
The current study provides several new insights in the understanding of the vascular effects of the ;1-adrenoreceptor antagonist nebivolol and a characterization of the underlying mechanisms: (1) nebivolol exerts a potent endothelium-dependent vasodilation of human and rodent coronary resistance arteries, as well as proangiogenic effects in vitro; (2) these effects are mediated through the stimulation of ;3-adrenoreceptors and NO production; and (3) these effects are accompanied by a rise in cytosolic calcium and an activating dephosphorylation of eNOS on Thr495 in ECs.
Whereas previous studies have shown a vasodilating effect of nebivolol in the human brachial vascular bed in vivo,7–9,20 our results demonstrate for the first time a very potent vasodilating effect of the drug in human coronary microvessels (70 to 170 μm in diameter), a major site of regulation of coronary resistance and perfusion reserve. This is of potential therapeutic importance, given the prognostic value of coronary microvascular dysfunction in human cardiomyopathies.21 As in other vascular beds,3,7,8 this effect is dependent on the presence of a functional endothelium and is sensitive to NOS inhibitors, but beyond previous studies, we now provide direct demonstration of NO release from ECs treated with nebivolol by 2 different techniques (Figure 4A and 4B).
Treatment of rat and human coronary microarteries with nadolol (a ;1-2-adrenoreceptor–specific blocker) did not significantly attenuate the relaxation to nebivolol, confirming its independence from the drug’s binding on ;1- or ;2-adrenoreceptors. Consistent with the results of Trochu and collaborators11 in the rat aorta, our previous study14 demonstrated that in human coronary microarteries, norepinephrine and a ;3-adrenoreceptor–preferential agonist, BRL 37344, evoked a sustained vasodilation that was partly mediated by NO. This was confirmed by the demonstration of specific immunohistochemical staining for ;3-adrenoreceptor proteins on the endothelium of the same vessels. We now show that nebivolol produces its vasodilation in similar preparations through interaction with this ;3-adrenoreceptor isotype. Indeed, contrary to the neutral effect of ;1-2-blockade in human (Figure 1D) and rodent (Figure 2A) vessels, the complete ;1-2-3-blocker bupranolol did significantly inhibit the relaxation to nebivolol in rat coronary microarteries (Figure 2A). Again, these results were fully consistent with the direct endothelial response to nebivolol in vitro (Figure 4C). Finally, the involvement of this ;3-isotype in nebivolol’s effect was definitively demonstrated by the impaired relaxation in aortas from mice genetically deficient in ;3-adrenoreceptors (Figure 2B). The identification of partial agonism on ;3-adrenoreceptors provides new mechanistic insights into the pharmacodynamic effect of this third-generation ;1-adrenoreceptor blocker, with coupling to critical ancillary effects on the vasculature.
NO is recognized as a major downstream effector of proangiogenic cytokines, such as VEGF, in ECs.22 Because we directly observed increased NO production upon stimulation with nebivolol in ECs, we tested whether the drug would also promote their adoption of a proangiogenic phenotype in vitro. We demonstrated that nebivolol induced microtube formation in isolated aortic rings, indicating proangiogenic properties (Figure 3). Our comparative use of rings from ;3-AR–/– and eNOS–/– mice again confirmed that this property depends on the presence of both eNOS and ;3-adrenoreceptors (Figure 3C and 3D). Although another study has shown that ;3-adrenoreceptor stimulation initiated proliferation and migration of ECs, 2 initial steps toward angiogenesis,15 we now directly demonstrate tube outgrowth from full, mature vessels clearly associated with ;3-adrenoreceptor stimulation by nebivolol.
Understanding of the molecular mechanisms of eNOS activation has recently progressed through the identification of regulatory phosphorylation sites that modulate eNOS activity (eg, Ser1177, Thr495) after increases in intracellular calcium. We previously demonstrated sequential events leading to eNOS activation on calcium-dependent dissociation of eNOS from caveolin-1, followed by recruitment of protein kinase Akt/PKB, resulting in phosphorylation of eNOS on Ser1177.17,23 The latter was strictly dependent on a rise in intracellular calcium concentration. Therefore, we examined the ability of nebivolol to interfere with several of these activation events. Our results demonstrate that when added to cultured ECs, nebivolol evoked a strong increase in fura-2 calcium signal (Figure 4C) of an amplitude similar to that of other endothelial agonists, such as bradykinin (not shown). However, we did not detect changes in phosphorylation of eNOS on Ser1177, suggesting that in our cells and experimental conditions, activation of eNOS by nebivolol does not involve phosphorylation of this particular residue. Conversely, we demonstrated that nebivolol induced dephosphorylation of the inhibitory phospho-Thr495 within the calcium-calmodulin consensus binding sequence of eNOS. In ECs activated with Ca2+-elevating agonists such as bradykinin (or nebivolol, as in our study), substantially more activating calmodulin binds to eNOS when Thr495 is dephosphorylated.24 Of note, this dephosphorylation on Thr495 was not accompanied by an increase in production of superoxide anions (as shown by insensitivity of the EPR signal for NO to a superoxide dismutase analogue; Figure 4B), as would have been anticipated from uncoupled eNOS.25
Potential Limitations and Pathophysiological Implications
Our results were obtained with nebivolol at a range of concentrations identical to those previously reported in the literature.3,26 Although it could be argued that concentrations used in vitro are higher than those achieved in patients, the effects of ;3-agonism presented here recapitulate the increases in human brachial blood flow7,9 observed at doses of nebivolol currently used therapeutically, which were also shown to be mediated by increases in NO production. Our data now suggest the possibility of similar effects in the human coronary vasculature. Broeders and collegues13 also demonstrated that sera from mice treated with nebivolol activated NO production in mouse aortic segments, suggesting the involvement of active metabolites, at least in mice. Although their paradigms indicated a ;2-adrenoreceptor effect, this was exclusively inferred from the use of butoxamine in mouse aortas. No other mouse or human vessel bed was examined.
The NO-releasing and vasodilating properties of nebivolol in coronary microvessels may underlie its beneficial effects in patients with ischemic or dilated cardiomyopathies,2 particularly those with diastolic dysfunction,27 given the direct lusitropic properties of NO in the myocardium.28 Diseases such as dyslipidemia, diabetes, and atherosclerosis are all associated with a decrease in eNOS expression, NO bioavailability, or both (due to, eg, oxidative stress), which could limit the clinical impact of a drug that depends on NO release. Importantly, in human coronary arterioles, we demonstrated that in situations where NO production is impaired, endothelium-derived hyperpolarizing factor(s) can fully compensate for the loss of NO and completely restore ;3-adrenoreceptor–mediated relaxation.14 This and the relative resistance of the ;3-adrenoreceptors to homologous desensitization29 would seem of particular importance in preserving myocardial perfusion in circumstances such as ischemia and heart failure. Finally, if reflective of similar properties in vivo, the proangiogenic effect of nebivolol would provide additional cardiovascular benefits in patients with ischemic cardiac or peripheral diseases.
In conclusion, our work demonstrates for the first time that nebivolol is able to elicit a potent and sustained relaxation of human coronary resistance arteries that is at least partially NO mediated. Our results obtained in both ECs and isolated coronary arteries substantiate the hypothesis that nebivolol exerts an agonist activity on ;3-adrenoreceptors to induce sustained NO production through increases in cytosolic calcium concentrations and dephosphorylation of Thr495-eNOS.
Acknowledgments
This work was supported by grants ARC 01/06-271 and PAI-P5/02 to Dr Balligand. Drs Dessy and Feron are research associates of the Fonds National de la Recherche Scientifique (FNRS); Julie Saliez and Fran;oise Frérart are recipients of a FRIA fellowship; Dr Belge and Géraldine Daneau are fellows of the FNRS. Part of this work was supported by Menarini Industrie, Firenze, Italy.
References
Van Lommen G, De Bruyn M, Schroven M. Synthesis and pharmacological properties of nebivolol, a new antihypertensive compound. J Pharm Belg. 1990; 45: 355–360.
Flather MD, Shibata MC, Coats AJ, Van Veldhuisen DJ, Parkhomenko A, Borbola J, Cohen-Solal A, Dumitrascu D, Ferrari R, Lechat P, Soler-Soler J, Tavazzi L, Spinarova L, Toman J, Bohm M, Anker SD, Thompson SG, Poole-Wilson PA. Randomized trial to determine the effect of nebivolol on mortality and cardiovascular hospital admission in elderly patients with heart failure (SENIORS). Eur Heart J. 2005; 26: 215–225.
Gao YS, Nagao T, Bond RA, Janssens WJ, Vanhoutte PM. Nebivolol induces endothelium-dependent relaxations of canine coronary arteries. J Cardiovasc Pharmacol. 1991; 17: 964–969.
Cosentino F, Bonetti S, Rehorik R, Eto M, Werner-Felmayer G, Volpe M, Luscher TF. Nitric-oxide-mediated relaxations in salt-induced hypertension: effect of chronic ;1-selective receptor blockade. J Hypertens. 2002; 20: 421–428.
Altwegg LA, d’Uscio LV, Barandier C, Cosentino F, Yang Z, Luscher TF. Nebivolol induces NO-mediated relaxations of rat small mesenteric but not of large elastic arteries. J Cardiovasc Pharmacol. 2000; 36: 316–320.
Ignarro LJ, Byrns RE, Trinh K, Sisodia M, Buga GM. Nebivolol: a selective ;1-adrenergic receptor antagonist that relaxes vascular smooth muscle by nitric oxide- and cyclic GMP-dependent mechanisms. Nitric Oxide. 2002; 7: 75–82.
Ritter JM. Nebivolol: endothelium-mediated vasodilating effect. J Cardiovasc Pharmacol. 2001; 38 (suppl 3): S13–S16.
Cockcroft JR, Chowienczyk PJ, Brett SE, Chen CP, Dupont AG, Van Nueten L, Wooding SJ, Ritter JM. Nebivolol vasodilates human forearm vasculature: evidence for an L-arginine/NO-dependent mechanism. J Pharmacol Exp Ther. 1995; 274: 1067–1071.
Dawes M, Brett SE, Chowienczyk PJ, Mant TG, Ritter JM. The vasodilator action of nebivolol in forearm vasculature of subjects with essential hypertension. Br J Clin Pharmacol. 1999; 48: 460–463.
Gray DW, Marshall I. Novel signal transduction pathway mediating endothelium-dependent ;-adrenoceptor vasorelaxation in rat thoracic aorta. Br J Pharmacol. 1992; 107: 684–690.
Trochu JN, Leblais V, Rautureau Y, Beverelli F, Le Marec H, Berdeaux A, Gauthier C. ;3-Adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium-derived nitric oxide in rat thoracic aorta. Br J Pharmacol. 1999; 128: 69–76.
Gosgnach W, Boixel C, Nevo N, Poiraud T, Michel JB. Nebivolol induces calcium-independent signaling in endothelial cells by a possible ;-adrenergic pathway. J Cardiovasc Pharmacol. 2001; 38: 191–199.
Broeders MA, Doevendans PA, Bekkers BC, Bronsaer R, van Gorsel E, Heemskerk JW, Egbrink MG, van Breda E, Reneman RS, van Der Zee R. Nebivolol: a third-generation ;-blocker that augments vascular nitric oxide release: endothelial ;2-adrenergic receptor-mediated nitric oxide production. Circulation. 2000; 102: 677–684.
Dessy C, Moniotte S, Ghisdal P, Havaux X, Noirhomme P, Balligand JL. Endothelial ;3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization. Circulation. 2004; 110: 948–954.
Steinle JJ, Booz GW, Meininger CJ, Day JN, Granger HJ. ;3-adrenergic receptors regulate retinal endothelial cell migration and proliferation. J Biol Chem. 2003; 278: 20681–20686.
Susulic VS, Frederich RC, Lawitts J, Tozzo E, Kahn BB, Harper ME, Himms-Hagen J, Flier JS, Lowell BB. Targeted disruption of the ;3-adrenergic receptor gene. J Biol Chem. 1995; 270: 29483–29492.
Brouet A, Sonveaux P, Dessy C, Balligand JL, Feron O. Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells. J Biol Chem. 2001; 276: 32663–32669.
Vanin AF. Iron diethyldithiocarbamate as spin trap for nitric oxide detection. Methods Enzymol. 1999; 301: 269–279.
Kleschyov AL, Munzel T. Advanced spin trapping of vascular nitric oxide using colloid iron diethyldithiocarbamate. Methods Enzymol. 2002; 359: 42–51.
Kubli S, Feihl F, Waeber B. ;-Blockade with nebivolol enhances the acetylcholine-induced cutaneous vasodilation. Clin Pharmacol Ther. 2001; 69: 238–244.
Cecchi F, Olivotto I, Gistri R, Lorenzoni R, Chiriatti G, Camici PG. Coronary microvascular dysfunction and prognosis in hypertrophic cardiomyopathy. N Engl J Med. 2003; 349: 1027–1035.
Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997; 100: 3131–3139.
Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res. 2001; 89: 866–873.
Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: e68–e75.
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
Parenti A, Filippi S, Amerini S, Granger HJ, Fazzini A, Ledda F. Inositol phosphate metabolism and nitric-oxide synthase activity in endothelial cells are involved in the vasorelaxant activity of nebivolol. J Pharmacol Exp Ther. 2000; 292: 698–703.
De Cree J, Vanwelden H, Geukens H, Verhaegen H. The effect of nebivolol in patients with left ventricular diastolic dysfunction. Drug Invest. 1991; 3: 142–143.
Massion PB, Balligand JL. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol. 2003; 546: 63–75.
Liggett SB, Freedman NJ, Schwinn DA, Lefkowitz RJ. Structural basis for receptor subtype-specific regulation revealed by a chimeric ;3/;2-adrenergic receptor. Proc Natl Acad Sci U S A. 1993; 90: 3665–3669.(Chantal Dessy, PhD; Julie)