Nikolaos Tzemos, Pitt O. Lim and Thomas M. MacDonald
Double-Blind, Crossover Study
Nebivolol Reverses Endothelial Dysfunction in Essential Hypertension : A Randomized,
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Nebivolol Reverses Endothelial Dysfunction
in Essential Hypertension
A Randomized, Double-Blind, Crossover Study
Nikolaos Tzemos, MRCP; Pitt O. Lim, MRCP; Thomas M. MacDonald, MD, FRCP, FESC
Background—Vascular endothelial dysfunction may predict future atherosclerosis. Hence, an antihypertensive agent that
reverses endothelial dysfunction and lowers blood pressure might improve the prognosis of patients with hypertension.
We hypothesized that nebivolol, a vasodilating ?-blocker, could improve endothelial dysfunction. We tested this
hypothesis by comparing the effects of nebivolol and atenolol on endothelial function.
Methods and Results—Twelve hypertensive patients with a mean ambulatory blood pressure of 154?7/97?10 mm Hg
were randomized after a 2-week placebo run-in period (baseline) in a double-blind, crossover fashion to 8-week
treatment periods with either 5 mg of nebivolol with 2.5 mg of bendrofluazide or 50 mg of atenolol with 2.5 mg of
bendrofluazide. Forearm venous occlusion plethysmography and intra-arterial infusions of acetylcholine and NG-mono-
methyl-L-arginine (L-NMMA) were used to assess stimulated and basal endothelium-dependent nitric oxide release,
respectively. Sodium nitroprusside was used as an endothelium-independent control. Nebivolol/bendrofluazide and
atenolol/bendrofluazide each lowered the clinic blood pressure to the same extent (132?7/82?6 and 132?9/
83?8 mm Hg, respectively; P?0.001 from baseline). The vasodilatory response to acetylcholine was significantly
increased with nebivolol/bendrofluazide (maximum percentage change in forearm blood flow [mean?SEM],
435?27%, P?0.001) but not with atenolol/bendrofluazide. Similarly, the endothelium-dependent vasoconstrictive
response to L-NMMA was significantly improved only with nebivolol treatment (percentage change in forearm blood
flow, ?54?5%; P?0.001). The response to sodium nitroprusside was not different between treatments, suggesting that
the endothelium-independent pathway was unaffected.
Conclusions—Nebivolol/bendrofluazide increased both stimulated and basal endothelial nitric oxide release, whereas for
the same degree of blood pressure control, atenolol/bendrofluazide had no effect on nitric oxide bioactivity. Thus,
nebivolol may offer additional vascular protection in treating hypertension. (Circulation. 2001;104:511-514.)
Key Words: hypertension ? endothelium ? nitric oxide
increase the systemic blood pressure in susceptible individu-
als, thus giving rise to hypertension.1Interestingly but per-
haps not unexpectedly, angiographically normal epicardial
coronary arteries in hypertensives have been shown to have
endothelial dysfunction, an abnormality that is now known to
precede atherosclerosis.2This is especially relevant because
hypertension is a major risk factor for ischemic heart disease.
The link between endothelial dysfunction and coronary artery
disease is now well established.3Hence, reversing endothelial
dysfunction in hypertension is an attractive pharmacological
aim if the natural history of the hypertension disease process
is to be altered. One candidate drug is nebivolol, a selective
n impairment in nitric oxide (NO) bioactivity or endo-
thelial dysfunction involving resistance arteries may
?1-receptor blocker that has vasodilating properties (attribut-
able to its ability to increase NO bioactivity, as demonstrated
in animals and human volunteers).4,5To discover whether
nebivolol can reverse endothelial dysfunction, we conducted
a double-blind, crossover trial comparing the effects of
nebivolol and atenolol on vascular NO bioactivity in patients
with essential hypertension.
Study subjects were recruited from those attending the Tayside
specialist hypertension clinic. Exclusion criteria included secondary
hypertension, coronary artery disease, diabetes, hyperlipidemia (fast-
ing cholesterol ?5.8 mmol/L), renal impairment or other vascular
Received April 23, 2001; revision received June 7, 2001; accepted June 8, 2001.
From the Hypertension Research Centre, Department of Clinical Pharmacology and Therapeutics, Ninewells Hospital and Medical School, Dundee
(N.T., T.M.M.), and the Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff (P.O.L.), United
A. Menarini Pharmaceuticals UK Ltd, which makes Nebivolol, provided the Hypertension Research Centre, where the authors are affiliated, with an
educational grant, part of which was used to fund this study.
Correspondence to Dr Nikolaos Tzemos, Hypertension Research Centre, Department of Clinical Pharmacology and Therapeutics, University of
Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom, DD1 9SY. E-mail firstname.lastname@example.org
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
Brief Rapid Communication
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diseases, and smoking. Written, informed consent was obtained from
each study subject. The Tayside committee on research medical
ethics approved this study.
This was a randomized, double-blind, crossover study. Suitable
study subjects had their antihypertensive drugs withdrawn at the
beginning of a 2-week placebo run-in period, at the end of which
each underwent 24-hour ambulatory blood pressure monitoring.
Subjects with daytime ambulatory blood pressures ?140 (systolic)
and/or ?90 (diastolic) mm Hg were then randomized to receive
either 5 mg of nebivolol with 2.5 mg of bendrofluazide per day or 50
mg of atenolol with 2.5 mg of bendrofluazide per day for 8 weeks.
Bendrofluazide (2.5 mg) was added at the beginning of each
treatment period to allow consistent blood pressure control in all
study subjects, rather than added later only for those in whom blood
pressure control was not achieved with monotherapy. At the end of
this first active treatment period, study subjects crossed over to the
alternate treatment arm for another 8 weeks after a further 2-week
placebo washout period.
Seated blood pressure was measured (mean of 3 measurements) at
the beginning of each visit after a 10-minute rest using a semiauto-
matic oscillometric monitor (OMRON 705CP).
Vascular endothelial studies were undertaken at the end of the first
2-week placebo run-in period to provide baseline data and at the end
of each 8-week active treatment period. Thus, each subject under-
went 3 endothelial function assessments. All studies were conducted
after an overnight fast and in a quiet, temperature-controlled labo-
ratory (24?0.5°C) with dimmed lights. Alcoholic and caffeinated
beverages were avoided for at least 24 hours before the study day.
After a supine rest of 30 minutes, the nondominant brachial artery
was cannulated with a 27-gauge steel needle mounted onto a
16-gauge polyethylene epidural catheter under local anesthesia with
1% lidocaine. Forearm blood flow (FBF) was measured simulta-
neously in both arms by strain-gauge venous occlusion plethysmog-
raphy, as previously described.6Blood pressure and heart rate were
noninvasively (OMRON, HEM-705CP) recorded in the noninfused
(control) arm before each infusion.
Hemodynamic Measurements and Drug Infusions
FBF was measured during the last 2 minutes of each infusion period
and was expressed as mL/100 mL forearm tissue/min, according to
the Whitney method.7Resting baseline FBF was obtained at least 30
minutes after needle placement to ensure that the blood flow in the
cannulated arm had stabilized. After resting baseline FBF measure-
ments, each study subject received intra-arterial infusions of incre-
mental doses of acetylcholine (Miochol, CIBAVision), sodium
nitroprusside (David Bull Laboratories), and NG-monomethyl-L-
arginine (L-NMMA; Clinalfa). The muscarinic agonist acetylcholine
was used to assess endothelium-dependent vasodilatation, whereas
sodium nitroprusside, an exogenous source of NO, was used to
assess endothelium-independent vasodilatation.
Cumulative dose response curves were constructed after infusions
of 25, 50, and 100 nmol/min acetylcholine and 4.2, 12.6, and 37.8
nmol/min sodium nitroprusside (each infusion lasted 5 minutes).
Endothelial-dependent vasoconstriction was assessed by using the
competitive NO synthase antagonist L-NMMA infused at 1, 2, and 4
?mol/min for 5 minutes. After each infusion, care was taken for FBF
to reach baseline values; this generally took at least 30 minutes. The
order of the vasoactive drugs infused was identical at all the study
visits, and the study subjects were unaware of the substances infused.
Drugs, saline, and 5% dextrose were infused at flow rates of 1
mL/min by means of a constant-rate infusion pump (Braun).
The FBF ratio between the infused and control arms in response to
drugs was expressed as a percentage of the ratio measured during the
baseline control period (?FBF%). The coefficient of variation of
FBF ranged from 7.8% to 16% (mean, 10.5%) when FBF was
analyzed repeatedly in a steady state. From previous studies, the
sample size was estimated with a power of 90% to detect a
cumulative ?FBF% difference between treatments of 100%. Clinical
characteristics between clinic visits were compared by paired Stu-
dent’s t test, and FBF measurements for individual treatments were
compared between treatments using 2-way ANOVA with repeated
measures with a correction for multiple comparisons for within-
group effects. A 2-tailed P?0.05 was considered significant. FBF
was expressed as mean?SEM; other values are mean?SD.
Twelve subjects (10 men and 2 postmenopausal women) with
a mean age of 52?7 years and long-standing (?5 years)
essential hypertension were studied. Daytime ambulatory
blood pressure monitoring at the end of the 2-week placebo
run-in period confirmed mild-to-moderate hypertension (Ta-
ble). Both nebivolol/bendrofluazide and atenolol/bendroflua-
Parameter Placebo (Baseline)NebivololAtenolol
ABP (daytime), mm Hg
Office SBP, mm Hg
Office DBP, mm Hg
Heart rate, beats/min
Absolute basal FBF,
mL ? 100 mL?1? min?1
Serum potassium, mmol/L
Serum creatinine, ?mol/L
? ? ?? ? ?
5.2?0.7/1.3?0.3 5.3?0.5/1.2?0.2 5.4?0.6/1.2?0.2
Values are mean?SD for all measurement except FBF, which is expressed as mean?SEM. ABP
indicates ambulatory blood pressure; SBP, systolic blood pressure; and DBP, diastolic blood pressure.
*P?0.05 and †P?0.001 for differences between treatments.
July 31, 2001
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zide treatments reduced systolic and diastolic blood pressures
to the same extent at the end of the each 8-week treatment
period compared with placebo (132?7/82?6 and 132?9/
83?8 versus 154?8/98?9 mm Hg, respectively; P?0.001).
However, office blood pressure readings were always ob-
tained early in the morning; therefore, we cannot exclude
significant blood pressure discrepancies from daytime to
night-time between treatment periods. Both ?-blocker/diuret-
ic treatments were well tolerated, and there were no signifi-
cant differences in baseline plasma electrolytes, urate levels,
or fasting cholesterol levels between the 3 study periods.
Blood pressure and absolute basal FBF (Table) were not
significantly different between the placebo lead-in period at
baseline and the washout period between the 2 active treat-
ments, and there were no significant carry-over effects.
Nebivolol/bendrofluazide treatment produced a significant
increase in forearm vasodilatation to acetylcholine compared
185?39%; P?0.001 for the difference between the whole
dose-response curves; see Figure). In contrast, atenolol/
bendrofluazide had no effect on acetylcholine-mediated (NO-
dependent) vasodilation. Neither nebivolol- nor atenolol-
based treatments significantly affected sodium nitroprusside–
induced vasodilation, which suggests that the endothelium-
independent pathway was unaffected. Only nebivolol-based
treatment improved the vasoconstrictive response to
L-NMMA compared with baseline (?FBF%, ?54?5% ver-
sus ?26?4%, respectively; P?0.001), which suggests an
additional and significant improvement of basal (tonic) NO
The present study demonstrated that nebivolol/bendroflua-
zide treatment lowered blood pressure and reversed endothe-
lial dysfunction in patients with essential hypertension.
Atenolol/bendrofluazide treatment similarly lowered blood
pressure but did not alter endothelial function.
Previous in vivo and in vitro studies have suggested that
nebivolol-mediated vasodilation is predominately endotheli-
um-dependent and favorably affects the L-arginine/NO path-
way.4,5In humans, nebivolol evokes endothelial-dependent
vasodilation in healthy volunteers and in hypertensives.5,8
However, these were short-term studies involving intrabra-
chial administration of the drug. Whether the same favorable
effects can be expected when given orally has not previously
been studied but would be of much greater relevance in
The precise mechanism by which nebivolol enhances NO
bioavailability is unclear, but the drug was recently shown to
increase phospholipase C activity, which increases intracel-
lular free calcium concentrations.9Because the activity of the
constitutive endothelial NO synthase is calcium/calmodulin-
dependent, an increase in the intracellular free calcium
concentration will activate this enzyme, with resultant in-
creases in NO release.10However, the endothelial cell surface
receptor that nebivolol acts on to mediate phospholipase C
activation has not yet been identified, although it could be a
serotonin (5-HT1) receptor. Kakoki and colleagues11showed
that 5-HT1blockade with a specific antagonist almost abol-
ished the vasodilatory response to nebivolol in a rat kidney
preparation. This suggests that some degree of pharmacolog-
ical cross-reactivity between serotonin- and ?-receptors does
occur, which may explain the vascular effects of nebivolol.12
Also, a possible antioxidant property of nebivolol has been
suggested as an additional factor in increasing NO bioactivity
or even reducing endothelin release.13,14In the present study,
we could not tell if the improvement in NO bioavailability
was secondary to an increased synthesis and/or reduced
deactivation of NO. However, the improvement of NO
bioactivity seen in the study may well explain some of the
properties of nebivolol that are not shared by other
Epidemiological studies have consistently shown that the
dominant effect of blood pressure lowering is that of stroke
reduction; the effect on reducing coronary artery disease has
been disproportionately less in hypertension.16This could be
because hypertension is only one of many cardiovascular risk
factors; thus, reducing blood pressure in itself may not be
sufficient to reduce mortality and morbidity. Pharmacological
treatment that reduces blood pressure and also reverses
endothelial dysfunction may have an additional impact on
reducing the incidence of cardiovascular events.
Percentage changes in FBF from baseline preceding each drug
infusion for 3 dose levels of acetycholine, sodium nitroprusside,
and L-NMMA after placebo (?), nebivolol (?), and atenolol (?)
therapy. Values are mean?SEM. *P?0.05 and **P?0.001 for
differences between treatments.
Tzemos et al Nebivolol Reverses Endothelial Dysfunction
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In conclusion, nebivolol/bendrofluazide but not atenolol/ Download full-text
bendrofluazide–based treatment lowered office (daytime)
blood pressure and reversed endothelial dysfunction in hy-
pertensive patients. Its use as an antihypertensive agent might
reduce the future atherosclerotic burden in hypertension and,
hence, could reduce the risk for cardiac events over and above
that expected by lowering blood pressure alone.
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