© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Coronary Endothelial Function Is Preserved With Chronic Endothelin Receptor Antagonism in Experimental Hypercholesterolemia In Vitro
From the Department of Internal Medicine, Division of Cardiovascular Diseases (P.J.M.B., D.R., D.R.H., A.L.), the Division of Hypertension (L.O.L.), and the Department of Physiology and Biophysics (J.C.R.), Mayo Clinic and Mayo Foundation, Rochester, Minn.
Correspondence to Amir Lerman, MD, Department of Internal Medicine and Cardiovascular Diseases, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail lerman.amir{at}mayo.edu
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Abstract—Hypercholesterolemia is associated with increased circulating and tissue endothelin-1 immunoreactivity, decreased nitric oxide (NO) activity, and altered endothelial function. We tested the hypothesis that chronic endothelin receptor antagonism preserves endothelial function and increases NO in experimental porcine hypercholesterolemia. Pigs were randomized to 3 groups: Group 1, a 2% high-cholesterol (HC) diet alone (n=7); group 2, RO-48-5695, a combined endothelin receptor antagonist, and an HC diet (n=8); and group 3, ABT-627, a selective endothelin-A receptor antagonist, and an HC diet (n=8). Coronary epicardial and arteriolar endothelial function was determined by a dose-response relaxation to bradykinin (10-11 to 10-6 mol/L), in all groups and in pigs maintained on a normal diet. Plasma total oxidized products of NO (NOx) were determined by chemiluminescence at baseline and after 12 weeks. Bradykinin-stimulated coronary epicardial and arteriolar relaxation in group 1 was attenuated compared with normal-diet controls. This relaxation was normalized with endothelin receptor antagonism. Plasma NOx decreased after 12 weeks in group 1 (-74.8±5.5%). This decrease was attenuated in the endothelin receptor antagonist groups (group 2, -28.2±15.0%; group 3, -38.9±20.6%). Chronic endothelin receptor antagonism preserves coronary endothelial function and increases NO in hypercholesterolemia. This study supports a role of endothelin-1 in the regulation of NO activity and suggests a possible therapeutic role for endothelin receptor antagonists in hypercholesterolemia.
Key Words: coronary vessels • hypercholesterolemia • endothelin receptors • nitric oxide • oxidative stress
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Introduction |
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Endothelin-1 is a 21–amino acid peptide that is atherogenic and has both mitogenic and vasoconstricting properties.1 2 3 These effects are exerted through the 2 endothelin receptors: the endothelin-A (ET-A) receptor located on vascular smooth muscle cells, and the endothelin-B (ET-B) receptor, located on both endothelial and vascular smooth muscle cells.4 5 Both receptors may mediate enhanced coronary vasoconstriction to endothelin-1 in pathophysiological states such as experimental hypercholesterolemia, in which levels of coronary tissue and circulating endothelin-1 are increased.6 7 8
Besides enhanced endothelin-1 activity, hypercholesterolemia is associated with abnormal coronary endothelial function, decreased basal nitric oxide (NO) activity, and decreased coronary endothelial cell NO synthase (NOS).6 9 Attenuation in NO activity is functionally important, since NO contributes to both basal and demand-mediated coronary blood flow, antagonizes the vascular effects of endothelin-1, and is intimately linked to the regulation of endothelin-1 production.10 11
Experimental hypercholesterolemia is also associated with increased production of oxygen free radicals and increased oxidative stress.12 13 This may subsequently lead to altered bioavailability of NO or functional changes of the endothelium and has important implications in the pathogenesis of atherosclerosis.14 15 16 Oxidative stress also increases endothelin-1 production and release from endothelial cells in vitro.17 18 Additionally, endothelin-1 may increase oxidative stress by increasing oxygen free-radical formation from macrophages.19
Therefore, the current investigation was undertaken with the objectives to determine (1) whether chronic endothelin receptor antagonism in hypercholesterolemia preserves coronary epicardial and arteriolar endothelial function in vitro, (2) whether chronic endothelin receptor antagonism attenuates the reduction in plasma total oxidized products of NO (NOx) and coronary endothelial NOS immunostaining associated with hypercholesterolemia, and (3) whether chronic endothelin receptor antagonism decreases plasma F2-isoprostane levels, an in vivo marker of oxidative stress, in experimental hypercholesterolemia.
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Animals
All study procedures with animals were reviewed and approved by the Mayo Foundation Institutional Animal Care and Use Committee and were designed in accordance with National Institutes of Health guidelines. Female juvenile, domestic, crossbred pigs (23 to 35 kg) (Larson Farms, Seargent, Minn) were placed on an atherogenic diet of 2% cholesterol and 15% lard by weight (TD 93296, Harlan Teklad) for 12 weeks.8 9 In the interim, group 1 animals (control group) did not receive any additional medications. Group 2 animals were placed on oral RO-48-5695 (Hoffmann–La Roche Ltd, Basel, Switzerland), a combined ET-A and ET-B receptor antagonist, on a weight-adjusted scale every 3 weeks to maintain the dose at 3 mg · kg-1 · d-1.20 The dosage of RO-48-5695 was determined on the basis of preliminary studies by Hoffmann–La Roche. Group 3 animals (n=8) were placed on ABT-627 (Abbott Laboratories, Abbott Park, Ill) on a weight-adjusted scale to maintain a dose of 4 mg · kg-1 · d-1.21 The dosage of ABT-627 was based on our previous studies.22 At the start of the study and after 12 weeks of therapy, animals were anesthetized with ketamine and xylazine, and the external carotid artery was exposed by cutdown and cannulated with an 8F arterial sheath for blood pressure measurement.
Plasma lipid profiles (Roche) were determined at baseline and after 12 weeks of the high-cholesterol diet.23 Additionally, plasma was obtained at baseline and after 12 weeks of the high-cholesterol diet in all 3 groups for determination of plasma F2-isoprostanes and NOx. Euthanasia was performed by intravenous administration of 30 mg/kg pentobarbital sodium (Sleepaway, Fort Dodge Laboratories) for the in vitro studies. Additional control animals maintained on a normal diet were used for the in vitro studies to determine normal vascular reactivity for comparison with the 3 groups on the high-cholesterol diet.
In Vitro Endothelial Function
After euthanasia, normal hearts as well as hearts from the
experimental animals were harvested for in vitro analysis of
coronary epicardial relaxation in response to bradykinin.
Epicardial Vessels
As previously described,24 the hearts were placed
into cold, modified Krebs-Ringer bicarbonate solution (control
solution) of the following millimolar composition: 118.3 NaCl, 4.7 KCl,
1.2 MgSO4, 1.2
KH2PO4, 2.5
CaCl2, 25.0 NaHCO3, 0.016
Ca-EDTA, and 11.1 glucose. Rings of tissue 2 to 3 mm long were
dissected from the left circumflex artery, transferred to organ
chambers with 25 mL of control solution (37°C, pH 7.4), and
oxygenated with 94% O2 and 6%
CO2. The tissue was suspended between 2 stirrups
and connected to a strain gauge for continuous recording of
isometric tension. The artery rings were equilibrated for 1 hour at a
resting tension. Viability of the vessels was confirmed by a
contractile response to 20 mmol/L KCl at baseline at 2, 4, and
6 g each time after the KCl had been washed out. At 6 g, all
vessels were exposed to substance P (10-6
mol/L, Sigma Chemical Co), an endothelium-dependent
vasodilator, to verify the functional integrity of the vascular
endothelium. All chambers were then washed out with the
control solution.
After an equilibrium period of 30 minutes, the rings were precontracted with 10-7 mol/L endothelin-1 (Phoenix Pharmaceuticals), and then the response to 10-11 to 10-6 mol/L bradykinin (Sigma) was recorded. Complete relaxation of each ring was obtained by exposure of the tissue to 10-3.5 mol/L papaverine (Sigma). In preliminary experiments, 10-7 mol/L endothelin-1 caused sustained contraction of porcine epicardial coronary arteries, and there was no difference in the contraction to endothelin-1 in any of the groups. Additionally, 10 rings from normal-diet pigs and 6 rings from group 1 pigs were preincubated with 10-4 mol/L NG-monomethyl-L-arginine (L-NMMA, Sigma), an NOS inhibitor, for 20 minutes before preincubation with endothelin-1, followed by the dose response to cumulative concentrations of bradykinin. In 9 rings from normal-diet pigs and 7 rings from group 1 pigs, the dose response to cumulative concentrations of endothelin-1 (10-10 to 10-7 mol/L) was also determined.
Arterioles
Coronary vasomotor tone in the arterioles was determined
by using previously described methods.7 25 Segments 2 to
3 mm long of the secondary branch of the left circumflex artery,
which were <500 µm in diameter, were dissected under a
dissecting microscope, transferred to an arteriograph filled with
control solution oxygenated with 96%
O2 and 4% CO2, and then
mounted onto microcannulas (Living System Instrumentation). Control
solution was circulated from a 250-mL oxygenated reservoir
through the arteriograph chamber at a flow rate of 12 mL/min.
Temperature was continuously monitored (model 7000H, Jenco Electronics)
to maintain the vessel environment at 37±0.5°C. The arteriograph was
placed on the stage of an inverted microscope (Diaphot-TMD, Nikon),
which had a video camera connected to the viewing tube. The signal
obtained from the video image of the vessel was processed by an
electronic system (Living System Instrumentation), which continuously
measured and recorded the inner diameter (lumen) and wall
thickness. Responses of the pressurized arteries were measured at a
transmural pressure of 50 mm Hg. The vessels were precontracted
with 10-8 mol/L endothelin-1, and then the
response to 10-11 to
10-6 mol/L bradykinin was recorded.
Complete relaxation of each arteriole was obtained by exposure to
10-4 mol/L papaverine (Sigma). Additionally, in
6 arterioles from normal-diet pigs, 10-4 mol/L
L-NMMA was added 20 minutes before the addition of
endothelin-1, followed by the dose response to cumulative
concentrations of bradykinin.
Plasma Total NOx
Plasma NOx levels were determined by
chemiluminescence with a Sievers nitric oxide analyzer (model
280) as previously described.26 27 Blood samples from each
group at baseline and after 12 weeks of therapy (group 1 n=7, group 2
n=8, and group 3 n=8) were centrifuged at 2500 rpm for 20
minutes at 10°C. The supernatant was removed and stored at -70°C.
The NO assay was standardized by a calibration curve by using known
concentrations of nitrate (0.01 µmol/L to 100 µmol/L)
obtained from NaNO3. For each sample, 4 µL of
sample was placed in a reducing vessel with 5 mL of 0.1 mol/L
vanadium(III)chloride, 1 mol/L HCl, and 100 µL of antifoaming agent
(Sievers) at 90°C. Each sample and standard were analyzed at
least 3 times. The mean value was used for all subsequent
analysis.
Endothelial NOS Immunostaining
Frozen sections of tissue were cut in 5-µm sections and
mounted on positively charged slides. The slides were dried at 37°C
for 1 hour and fixed in acetone for 10 minutes at 4°C. The slides
were air dried for 30 minutes. Endogenous peroxidase
activity was blocked by placing the slides in 1.5%
H2O2 and 50% absolute
methanol for 10 minutes and then rinsed. The slides were pretreated
with 0.25% SDS for 10 minutes. To block nonspecific binding sites, the
tissue was incubated with 5% goat serum (Dako)/PBS/Tween 20 for 10
minutes. Monoclonal antibodies to endothelial NOS
(Transduction Laboratories) were then added (400 µL of a 0.25 µg/mL
dilution) and incubated overnight at 4°C. The slides were rinsed,
incubated for 30 minutes with biotinylated goat anti-mouse IgG diluted
1:400 and 2% normal swine serum, rinsed, incubated with
peroxidase-labeled streptavidin diluted 1:500 for 30 minutes, and
rinsed. Next, the tissue was stained for 15 minutes with 3-amino-9
ethylcarbazole solution and rinsed. To enhance nuclear detail, the
slides were counterstained with hematoxylin. Three independent
observers who were blinded to the different treatment groups reviewed
the tissue sections. The presence of staining was quantified on the
basis of a scoring system as previously described6 : 0, no
staining; 1, positive staining in <25% of the
endothelial cells per slide; 2, positive staining in
25% to 75% of the endothelial cells; and 3, positive
staining in >75% of the endothelial cells. The scores
for each of the observers were averaged for each of the slides, and the
average score for each of the groups was then calculated.
F2-Isoprostanes
Blood samples from 5 pigs in each of the 3 groups at baseline
and after 12 weeks of therapy were collected in EDTA tubes, and the
plasma was stored at -80°C until the time of the assay. The total
levels of 8-isoprostaglandin F2 were
measured with an enzyme immunoassay kit (EIA, Cayman). Before the
enzyme immunoassay, alkaline hydrolysis was used.28 Plasma
samples were purified by Sep-Pak C-18 columns (Waters) before
analysis. The samples, tracer, and antiserum were added to
wells precoated with mouse monoclonal antibody. The plates were washed
to remove all unbound reagents. Ellman’s reagent (containing the
substrate to acetylcholinesterase) was added to the wells. The
intensity of the distinct yellow color produced by this enzymatic
reaction was determined with a spectrophotometer at 405 nm.
Statistical Analysis
Data are expressed as mean±SEM. Within each group, repeated
measurements were analyzed with repeated-measures ANOVA
followed by the Bonferroni t test or by Student’s paired
t test, unpaired t test between groups, or the
Mann-Whitney rank-sum test. Statistical significance was achieved with
a value of P<0.05.
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Results |
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Control Group
After 12 weeks of the high-cholesterol diet, total cholesterol significantly increased (the Table
). This was associated with a
significant increase in HDL and LDL, whereas triglycerides
did not change significantly. In this group after 12 weeks, there was
an increase in mean arterial blood pressure (99±6
mm Hg versus 134±5 mm Hg, P<0.05). This increase in
mean arterial blood pressure occurs with the increasing
size of the animal and regardless of whether the pigs were fed the
normal diet or the high-cholesterol
diet.6
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In epicardial coronary vessels removed from the
hypercholesterolemic pigs, the in vitro response to
bradykinin was attenuated compared with that in the normal-diet pigs
(maximal relaxation 70.80±6.22% versus 88.66±2.26%, respectively;
P<0.05; Figure 1). There was
no difference in the epicardial vessels from the
high-cholesterol diet pigs compared with vessels from
normal-diet pigs in their response to endothelin-1 (normal endothelin-1
precontraction 11.09±0.79 g tension, group 1 endothelin-1
precontraction 8.67±0.86 g tension; P=0.06) or KCl (data
not shown). Additionally, preincubation of the vessels with
L-NMMA did not alter the magnitude of the
precontraction with endothelin-1 (with L-NMMA
9.62±1.37 g tension versus without L-NMMA
11.09±0.79 g tension; P=0.42). The
endothelium-dependent coronary vasorelaxation
in response to the maximal dose of bradykinin
(10-6 mol/L) was significantly attenuated in
the hypercholesterolemic, porcine epicardial vessels
(n=9 rings) compared with the normal vessels (n=7 rings).
L-NMMA significantly attenuated the response to
bradykinin in the normal vessels (maximal relaxation: 38.66±6.47%
with L-NMMA versus 88.66±2.26% without
L-NMMA; P<0.001) and in the
high-cholesterol vessels (maximal relaxation:
59.78±13.49% with L-NMMA versus 70.80±6.22%
without L-NMMA; P=0.022).
Endothelium-independent vasorelaxation to sodium
nitroprusside (10-9 to
10-4 mol/L) was not attenuated with the
high-cholesterol diet (maximal relaxation: normal diet
75.79±4.54%, group 1 75.00±6.78%). The response of the epicardial
vessels to cumulative concentrations of endothelin-1 was not altered in
rings from pigs fed the high-cholesterol diet compared with
the control rings from pigs on the normal diet (maximal contraction
compared with 60 mmol/L KCl in the normal vessels:
131.14±12.84%, group 1: 116.78±9.01%; P=0.378). The in
vitro arteriolar response to bradykinin at the higher concentrations
(10-7.5 to 10-6 mol/L,
Figure 2) similarly showed an attenuated
endothelium-dependent relaxation in the
high-cholesterol pigs (n=6 rings) compared with the
normal-diet pigs (n=7 rings). The arteriolar response to bradykinin was
again significantly attenuated with the addition of
L-NMMA in arterioles from normal-diet pigs
(78.42±4.08% with L-NMMA versus 96.57±1.34%
without L-NMMA; P<0.001).
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In group 1, plasma NOx values significantly
decreased after 12 weeks of the high-cholesterol diet
compared with baseline (P=0.01; the Table
). This
decrement was associated with an overall decrease in immunoreactivity
for NOS in coronary vessels after 12 weeks of the
high-cholesterol diet compared with pigs on the normal diet
(2.8±0.1 versus 1.2±0.4; P<0.01; Figure 3).
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Plasma F2-isoprostane levels significantly
increased in the control group after 12 weeks of the
high-cholesterol diet (the Table). In previous
studies in our laboratory, normal-diet pigs have been shown to not have
alterations in plasma F2-isoprostane levels after
12 weeks.
Endothelin Receptor Antagonist Groups
After 12 weeks of the high-cholesterol diet, total
cholesterol significantly increased in both of the
endothelin receptor antagonist groups compared with
baseline. There was no difference in cholesterol levels
(total cholesterol, HDL, or LDL) between the 3 groups at
baseline and after 12 weeks. Additionally, the lipid profile in the
normal-diet control group used for the organ chamber experiments was
similar to all 3 groups at baseline (total cholesterol
83.6±7.15 mg/dL). In both group 2, treated with the combined ET-A/ET-B
receptor antagonist, and group 3, treated with the
selective ET-A receptor antagonist, the normal increase in
blood pressure seen in the control group was attenuated (104±6
mm Hg and 108±2 mm Hg, respectively, compared with 134±5
mm Hg in group 1).
There was no difference in the response of the epicardial vessels in
any of the groups to endothelin-1 (group 2 endothelin-1 precontraction
8.40±1.48 g tension; P=0.87 versus group 1; group 3
endothelin-1 precontraction 11.33±0.98 g tension; P=0.07
versus group 1) or KCl (data not shown). The attenuated
endothelium-dependent vasorelaxation of the
hypercholesterolemic epicardial vessels was normalized
when either the combined ET-A/ET-B receptor antagonist
(n=6, group 2) or the selective ET-A receptor antagonist
(n=6, group 3) (maximal relaxation 93.25±4.94% and 86.50±4.67%,
respectively) was given chronically in combination with the
high-cholesterol diet (Figure 1). The in vitro
arteriolar studies (mean size of the vessel was 392±17 µm)
showed a similar trend as observed in the epicardial vessels. The
attenuated endothelium-dependent relaxation to
bradykinin in the high-cholesterol pigs was normalized in
both the combined ET-A/ET-B receptor antagonist group (n=8)
and in the selective ET-A receptor antagonist group (n=7,
Figure 2).
After 12 weeks, the decrease in plasma NOx
observed in the control group was significantly attenuated in both
group 2 and group 3 (Figure 4).
Similarly, there was an increase in the presence of enzyme
immunoreactivity for endothelial NOS in the
coronary arteries obtained from the endothelin
antagonist groups (groups 2 and 3, 2.1±0.3 and 2.3±0.3)
compared with the group on the high-cholesterol diet alone
(group 1, 1.2±0.4; Figure 3).
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Compared with group 1, chronic endothelin receptor antagonism
also significantly attenuated the increase in plasma
F2-isoprostanes from baseline in both the
combined endothelin receptor antagonist group, from
108.0±9.7 to 158.4±15.9 pg/mL at 12 weeks, and the selective ET-A
receptor antagonist group, from 110.4±5.0 to 154.4±14.4
pg/mL (Figure 5).
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The current study demonstrates that chronic endothelin receptor antagonism in porcine experimental hypercholesterolemia, by either the combined ET-A/ET-B receptor antagonist or the selective ET-A receptor antagonist, normalizes coronary epicardial and arteriolar endothelial function. This was associated with increased plasma NOx and increased coronary immunoreactivity for constitutive NOS. Finally, these data show that chronic endothelin receptor antagonism attenuates the increase in F2-isoprostanes in porcine hypercholesterolemia. This study supports a role for endothelin in the progression of coronary atherosclerosis in experimental porcine hypercholesterolemia.
Endothelin-1 is a potent vasoconstrictor and atherogenic peptide with enhanced immunoreactivity in atherosclerotic tissue.1 29 30 Furthermore, increased endothelin-1 is associated with most atherosclerotic risk factors.8 31 Thus, this peptide may be part of a common mechanism in the evolution of coronary atherosclerosis. In atherosclerosis, endothelin-1 alters vascular remodeling by inhibiting apoptosis, promoting proliferation of smooth muscle cells and fibroblasts, and acting as a chemoattractant factor and activator of macrophages.32 33
In addition to its direct atherosclerotic effects, endothelin-1 may in part mediate its effects through decreased NO production. NO antagonizes both the atherogenic and vasoconstricting effects of endothelin-1.34 35 Furthermore, the regulatory mechanisms of these vasoactive factors interact. NO decreases both the production and the release of endothelin-1 from the vascular endothelium.36 37 Endothelin-1 in turn modulates NO production through inhibition of NOS.38 NO also modulates both the number and affinity of ET-A receptors.39 In disease states such as hypercholesterolemia and atherosclerosis, an imbalance between NO and endothelin-1 activity is detected and may contribute to both vasomotor abnormalities and vascular remodeling.1 Thus, determining the effect of chronic endothelin receptor antagonism on NO may demonstrate a mechanism for the antiatherosclerotic effect.
Our study demonstrates that in hypercholesterolemia, chronic endothelin receptor antagonism partly preserves the metabolic products of NO, as determined by total NOx, suggesting that the amount of NO produced is partly preserved. This was associated with an attenuated decrease in coronary endothelial NOS immunoreactivity and preserved coronary endothelial function. The response to bradykinin was similar between the high-cholesterol group and the normal group with L-NMMA, suggesting that the attenuated response in the high-cholesterol group may be due to decreased NO bioavailability in the epicardial vessels and arterioles. Thus, this suggests that preservation of bradykinin-stimulated vascular relaxation in the endothelin antagonist groups is also associated with preserved agonist-stimulated NO release. We have recently demonstrated that experimental hypercholesterolemia is characterized by enhanced coronary vasoconstriction to endothelin-1 in vivo without a change in endothelin receptor density.6 This was associated with a decrease in endogenous coronary NO activity.6 Therefore, preservation of NO production could potently attenuate the enhanced vasoconstrictor effects of endothelin-1. Furthermore, endothelial function has been proposed as a marker for early atherosclerosis.40 These data suggest that 1 of the mechanisms for the antiatherosclerotic effects of chronic endothelin receptor antagonism may be through the effects on NO.
Besides increased endothelin-1, experimental hypercholesterolemia is also associated with increased vascular production of oxygen free radicals. This leads to augmented oxidation of LDL, which appears to be pivotal in the formation and progression of atherosclerosis through multiple pathways, including enhanced cellular LDL uptake and proinflammatory effects.12 13 41 42 Oxidative stress also promotes atherosclerosis by increasing endothelin-1 production and inactivation of NO by oxygen free radicals.14 17 18 However, through the same scavenger mechanism, NO can inhibit lipid peroxidation and the formation of F2-isoprostanes.43 Moreover, endothelin may increase oxidative stress by increasing oxygen free-radical formation from macrophages.19 Thus, oxidative stress promotes an atherogenic environment of increased endothelin-1, decreased NO, and increased lipid peroxidation. In addition to the atherosclerotic effects, oxidative stress can also alter vascular tone. In part, this effect may be mediated through the alteration in the balance of NO and endothelin-1. Thus, oxidative stress increases the atherosclerotic process and increases vascular tone.
Our study demonstrates that in hypercholesterolemia, chronic inhibition of endothelin receptors by either combined ET-A/ET-B receptor antagonism or by selective ET-A receptor antagonism attenuates the increase in circulating F2-isoprostane concentrations, an in vivo marker of endogenous lipid peroxidation. This suggests that not only may oxidative stress alter endothelin-1 production but also that endothelin-1 may alter oxidative stress. Furthermore, decreased oxidative stress with subsequent increased NO bioavailability and decreased lipid peroxidation may be 1 of the mechanisms for the antiatherosclerotic effects of endothelin receptor antagonists. In addition, this study shows that decreased oxidative stress is associated with normalization of bradykinin-stimulated endothelium-dependent vasodilatation in vitro. This effect may in part be due to preservation of NO production and decreased formation of vasoconstricting substances, including F2-isoprostanes. Despite this potential mechanism, we cannot rule out the possibility that the chronic hemodynamic effects of endothelin receptor antagonism may in part mediate some of these effects. Chronic endothelin antagonists lower blood pressure in both normal-diet pigs and, in the current study, in pigs fed a high-cholesterol diet.22 The improvement in endothelial function seen with chronic endothelin antagonists is unlikely to be due to acute hemodynamic effects of endothelin receptor antagonism, since we have previously demonstrated that acute endothelin receptor antagonism does not attenuate hypercholesterolemia-induced endothelial dysfunction.9 Furthermore, this study demonstrated that acute endothelin receptor antagonism does not alter endothelial function in normal-diet pigs.
To determine the relative importance of each receptor type in the hypercholesterolemic state, this study used both a combined ET-A/ET-B receptor antagonist and a selective ET-A receptor antagonist. Because the ET-B receptor is functionally coupled to NOS, one could speculate that blockade of this receptor might be deleterious. However, we and others have previously demonstrated that in pathophysiological states, the ET-B receptor predominantly mediates vasoconstriction.7 44 45 In our current study, blockade of the ET-B receptor as part of the combined ET-A/ET-B receptor blockade showed similar effects as blockade of the ET-A receptor alone. Thus, this study did not show any added benefit of ET-B receptor antagonism in hypercholesterolemia.
In summary, the present study demonstrates that chronic endothelin receptor antagonism in experimental hypercholesterolemia can preserve coronary endothelial function, augment NO production, and decrease oxidative stress. These data support a role of endothelin-1 in the regulation of NO production and suggest a possible therapeutic role for endothelin receptor antagonists in pathophysiological states.
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Acknowledgments |
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This work was supported by National Institutes of Health training grant HL07111-21D (to P.B.), the Miami Heart Research Institute (to A.L.), the Bruce and Ruth Rappaport Program in Vascular Biology (to A.L.), and the Mayo Foundation (all authors). Additionally, we would like to thank Dr Terry J. Opgenorth from Abbott Laboratories for providing ABT-627, Dr Martine Clozel from Hoffmann–La Roche Ltd for providing RO-48-5695/005, and Paula Carlson for her technical support.
Received October 22, 1998; accepted March 22, 1999.
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