(Circulation. 1996;93:1009-1019.)
© 1996 American Heart Association, Inc.
Articles |
Effects of Subtype-Selective and Balanced Angiotensin II Receptor Antagonists in a Porcine Coronary Artery Model of Vascular Restenosis
From the Departments of Pharmacology (W.R.H., R.C.M., T.F., I.I.S., R.S.L.C., C.F.R., R.G.J.), Laboratory Animal Resources (M.D.D., W.R.A., M.P.), Biometrics (D.J.H.), and Exploratory Chemistry (D.K., D.L.O., N.B.M., W.J.G.), Merck Research Laboratories, West Point, Pa; and the Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic and Foundation, Rochester, Minn (R.S.S.).
Correspondence to Dr William R. Huckle, Merck Research Laboratories, WP42-300, West Point, PA 19486. E-mail huckle@merck.com.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Numerous studies have demonstrated the ability of angiotensin II (Ang II) receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors to inhibit intimal hyperplasia after balloon dilatation of noncoronary arteries in small-animal models, suggesting an important role for Ang II in the response to injury. Although ACE inhibitors have not been similarly effective in nonhuman coronary models or in human restenosis trials, questions remain regarding the efficacy of ACE inhibitors against tissue ACE and the contributions of ACE-independent pathways of Ang II generation. Unlike ACE inhibitors, Ang II receptor antagonists have the potential to inhibit responses to Ang II independent of its biosynthetic origin.
Methods and Results In separate studies, three Ang II receptor antagonists, including AT1-selective (L-158,809), balanced AT1/AT2 (L-163,082), and AT2-selective (L-164,282) agents, were evaluated for their ability to inhibit vascular intimal thickening in a porcine coronary artery model of vascular injury. Preliminary studies in a rat carotid artery model revealed that constant infusion of L-158,809 (0.3 or 1.0 mg·kg-1·d-1) reduced the neointimal cross-sectional area by up to 37% measured 14 days after balloon dilatation. In the porcine studies, animals were treated with vehicle or test compound beginning 2 days before and extending 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of commercially available angioplasty balloons with placement of coiled metallic stents. Infusion of L-158,809 (1 mg·kg-1·d-1), L-163,082 (1 mg·kg-1·d-1), or L-164,282 (1.5 mg·kg-1·d-1) in the study animals yielded plasma drug levels sufficient either to chronically block or, for L-164,282, to spare pressor responses to exogenous Ang II. Neither L-158,809, L-163,082, nor L-164,282 had statistically significant effects (P=.12, P=.75, and P=.48, respectively, compared with vehicle-treated controls) on neointimal thickness (normalized for degree of injury) measured by morphometric analysis at day 28 after angioplasty.
Conclusions These findings indicate that chronic blockade of Ang II receptors by either site-selective or balanced AT1/AT2 antagonists is insufficient to inhibit intimal hyperplasia after experimental coronary vascular injury in the pig. The results further suggest that, unlike in the rat carotid artery, Ang II is not a major mediator of intimal thickening in the pig coronary artery.
Key Words: angiotensin • restenosis • angioplasty • receptors • coronary disease
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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PTCA is used widely for the enhancement of arterial blood flow in occlusive coronary artery disease. The effectiveness of this procedure is limited by the development of vascular restenosis in 30% to 50% of individuals who undergo angioplasty with either balloon, atherectomy, or laser devices.1 Efforts to limit the occurrence of restenosis pharmacologically have examined a variety of antithrombotic, antiproliferative, anti-inflammatory, antihypertensive, and hypolipidemic agents. To date, however, no clinical studies using large sample sizes, adequate rates of follow-up, and strict angiographic criteria for diagnosing restenosis have successfully limited restenosis pharmacologically.2 3 The possible involvement of the renin-angiotensin system in restenosis has received considerable experimental scrutiny. Ang II, the major bioactive product of this system, is recognized to exert growth-promoting effects on a variety of target tissues, including the vasculature (reviewed in References 4 through 6). Using a rat carotid artery model of restenosis, Powell and colleagues7 demonstrated that the ACE inhibitor cilazapril could inhibit the formation of neointima after injury by balloon dilatation. Similar findings have been made in the rat carotid artery model with other ACE inhibitors, including captopril,8 ramipril,9 benazeprilat,10 and delapril.11 These findings, together with observations that exogenous Ang II can potentiate neointimal formation12 and that balloon injury enhances the local expression of angiotensinogen mRNA,13 ACE activity,14 15 and AT1 Ang II receptor binding activity,16 have reinforced the idea that Ang II may be an important mediator of neointimal hyperplasia.
Attempts to extend the results with ACE inhibitors to other species have met with mixed results. Cilazapril was found to inhibit neointimal formation in ballooned guinea pig carotid but not rabbit iliac arteries.17 In porcine models of restenosis involving injury to the coronary arteries, neither cilazapril,18 enalapril,19 trandolapril, nor captopril20 was effective. Likewise, cilazapril was ineffective in a baboon model of restenosis.21 Most significantly, human clinical trials with cilazapril (MERCATOR22 and MARCATOR23 ) or fosinopril24 have shown no beneficial effects on overall clinical outcome after PTCA.
Although these results do not support the hypothesis that Ang II is a
significant mediator of restenosis, questions remain as to
whether ACE inhibitors are adequate
physiological antagonists of Ang II in
this context (Fig 1
). The possibility exists, for
example, that ACE localized at the site of vascular injury may be
refractory to inhibition by antihypertensive doses of ACE
inhibitors.15 The potential importance of this
factor is highlighted by findings that ACE levels are markedly
increased in neointimal tissue25 and that
overexpression of ACE in rat carotid arteries produces a hypertrophic
response that can be inhibited by the AT1 Ang II receptor
antagonist losartan.26 In addition,
ACE-independent pathways of Ang II biosynthesis (eg, involving chymase
activities) have been detected in human but not rat
arteries,27 28 raising the possibility that, in
humans,
significant formation of Ang II might persist in the presence of ACE
inhibitors. Third, ACE inhibitors can
potentiate the effects of the vasodilator bradykinin by inhibiting its
degradation; in the rat carotid artery, part of the
inhibitory action of ACE inhibitors on
neointimal formation has been attributed to
kinins.29
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The heterogeneity of Ang II receptor subtypes
presents another complicating factor in the interpretation of ACE
inhibitor restenosis studies. Complete blockade
of Ang II synthesis would alter Ang II signaling through both
AT1 and AT2 receptor subtypes, with
unpredictable consequences. Occupancy of the AT2 site with
Ang II does not elicit the spectrum of responses associated with the
AT1 site (Fig
1
),30 31 32 33
and although the
AT2 site has been associated with a variety of cellular
responses,34 35 36 37 38 39 40
there is no well-accepted model of
AT2 signaling or physiology. Interest in the possible role
of the AT2 receptor in vascular injury has remained high,
given the high levels of AT2 expression in neonatal rat
aorta and its induction in adult rat carotid artery by balloon
injury.41 In addition, recent studies have shown that
overexpression of AT2 in the carotid artery has a
growth-inhibiting effect.42 Thus, inhibiting Ang II
synthesis may blunt both the growth-promoting and
growth-suppressing effects of Ang II in the vasculature.
The availability of specific, site-selective Ang II receptor antagonists has presented the opportunity to address these issues. In the rat carotid injury model, numerous studies have demonstrated the efficacy of AT1-selective compounds, including losartan10 11 43 44 45 46 47 and TCV-116,48 as well as the nonselective antagonists saralasin49 and [Sar1,Phe(Br5)8]Ang II.50 Interestingly, the AT2-selective agents PD12331947 and CGP 42112A45 also were reported to inhibit neointimal formation in the rat. In the present studies, we evaluated site-selective and balanced AT1/AT2 Ang II receptor antagonists as inhibitors of intimal thickening in a porcine coronary artery model of vascular restenosis. This model was chosen because the pig has a coronary size and vascular distribution similar to that of humans and develops a neointima that closely resembles human restenotic lesions grossly and microscopically.51 Moreover, degrees of injury and neointimal response are readily quantifiable in this model.52 Testing the effects of Ang II receptor blockade on neointimal formation has important clinical implications, given the number of Ang II antagonists now in the late phases of clinical development.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Ang II Receptor Ligands
Ang II (human sequence; Peninsula) was freshly prepared as a 0.1-mg/mL stock solution in saline. Synthesis of L-158,809 (Fig 2
), an AT1-selective Ang II receptor ligand
with no detectable agonist activity,53 has been
described.54 The potassium salt of L-158,809 was prepared
at 3 mg/mL in 15% saturated NaHCO3.
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Structural analogues of
L-163,082 (Fig 2) with similar AT1
and AT2 binding affinities but inferior in vivo
profiles, have been described.55 The synthesis of
L-163,082 is outlined in Fig 3. Nitration of
2-amino-4-methyl-3-nitropyridine ([1] in Fig 3)
provided
2-amino-4-methyl-3,5-dinitropyridine ([2] in Fig 3).
The nitro groups
were reduced by the action of H2 and Raney nickel catalysis
to yield 4-methyl-2,3,5-pyridinetriamine, which is unstable upon
exposure to atmospheric oxygen and was therefore isolated as its
hydrochloride salt [3]. Treatment of [3] with
n-PrCO2H in polyphosphoric acid resulted in
imidazopyridine formation with concomitant amidation to afford [4]
in
high yield. Alkylation of [4] with
4-bromomethyl-3-fluoro-2'-tert-butylamino-sulfonyl[1,1']biphenyl56
followed by deprotection with trifluoroacetic acid provided [5]. The
6-benzamide group was introduced by hydrolysis to the free amine [6]
followed by treatment with benzoyl chloride to yield [7]. Reaction
of
[7] with n-butylchloroformate afforded L-163,082 (Fig
2).
The potassium salt of L-163,082 was prepared at 0.4 mg/mL in a vehicle
containing 0.39% (wt/vol) NaCl, 5% saturated
NaHCO3, and 2% (wt/vol) Tween 20. The synthesis of
L-164,282 was similar to that described for L-163,082. L-164,282 was
prepared as a 2-mg/mL solution in 15% saturated NaHCO3.
All solutions of Ang II receptor ligands for intravenous
administration were filter-sterilized (0.2 µm) before use.
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In Vitro Methods
Vascular SMC Cultures
For
porcine coronary SMC cultures, the right, left
anterior descending, and left circumflex coronary arteries were
carefully dissected from the heart of a pig and flushed with cold PBS
containing penicillin and streptomycin. Fat and connective tissue were
trimmed from the exterior of each vessel. The medial layers were
extruded by applying pressure along the length of the vessel with a
pair of forceps, and the tissue recovered was finely minced with
scissors. Medial cells were dispersed by incubation in HBSS containing
3 mg/mL collagenase and 0.5 mg/mL elastase
(Worthington) with gentle agitation for 1 hour at 37°C. Dissociated
SMCs were recovered by centrifugation at
200g for 10 minutes. Cells were washed three times with HBSS
to remove residual proteases. The final SMC pellet was resuspended in
Waymouth's MB 752/1 medium (GIBCO) containing 5% fetal bovine serum,
1 mmol/L sodium pyruvate, 50 µg/mL gentamycin, and 50 ng/mL
amphotericin B. The suspension was cultured in a 25-cm2
flask at 37°C in a humidified atmosphere containing 5%
CO2 in air. The medium was changed after 24 hours and every
3 days thereafter. The primary SMC culture reached confluence after 7
to 10 days and was subcultured weekly at a split ratio of 1:5 in the
culture medium described above. Cells of passages 5 through 10 were
used for binding studies. Analogous procedures were used for the
preparation of rat aortic SMC cultures.
Ang II Receptor
Binding Assays and Determination of Plasma Levels
of Test Compounds
Levels of L-158,809 or L-163,082 in rat or pig
plasma were
estimated by radioreceptor assays using rat aortic SMC cultures
(passages 5 through 17) in which only the AT1 Ang II
receptor subtype is detectable (data not shown). Confluent cultures in
24-well culture dishes (Costar) were washed twice with 0.5 mL WBH.
Binding incubations (0.3 mL in WBH plus 10 U/mL heparin) contained 50
pmol/L [125I]Sar1Ile8Ang II plus
10% (vol/vol) test plasma or 10% control plasma spiked with known
concentrations of L-158,809 or L-163,082. After 60 minutes at 23°C,
radioligand-containing media were removed, and cells
were washed twice with 0.5 mL ice-cold WBH to remove unbound
ligand. Bound radioligand was solubilized with 0.5 mL 0.1
mol/L NaOH/1% SDS, and 125I was quantified by gamma
spectroscopy (model 1277, LKB-Wallac). The degree of inhibition of
radioligand binding in the presence of test plasma was
converted to receptor antagonist concentration by use of
standard curves linearized by the logit-log
transformation.57 Detection limits for L-158,809 and
L-163,082 by this method were 5 and 10 nmol/L, respectively. Results
from these assays represent estimates of total plasma
antagonist, ie, including that bound to plasma proteins,
since both standards and unknowns contained plasma. The same procedure
was used for assays of Ang II receptor antagonists on pig
SMC cultures, except that plasma and heparin were not present
during binding incubations.
Levels of L-164,282 were estimated by radioreceptor assay using bovine cerebellar membranes (Dupont-NEN) as a source of AT2 receptors. Plasma was incubated for 1 hour at 37°C with AT2-containing membranes plus 100 pmol/L [125I]Sar1Ile8Ang II (final plasma concentration, 10% vol/vol; total volume, 0.25 mL). Membranes were collected by aspiration onto a Skatron filter mat and washed with ice-cold 0.9% saline to remove unbound radioligand. The detection limit for L-164,282 was 3 nmol/L. For assays of binding to pig AT2 receptors, crude membrane fractions were prepared from porcine adrenal glands obtained from a local abattoir. Suspensions of membrane protein were incubated with 50 pmol/L [125I]Sar1Ile8Ang II and varying concentrations of Ang II receptor antagonists in the presence of 25 mmol/L Tris-HCl, 75 mmol/L NaCl, 2.5 mmol/L EDTA, and 0.02% BSA, pH 7.5, for 1 hour at 37°C. Bound radioligand was determined as described for bovine cerebellar membranes.
In Vivo Studies
Rat Carotid Angioplasty
All
animal procedures were performed according to protocols
approved by our institutional animal care and use committee. Balloon
dilatation of rat carotid arteries was performed essentially as
described.58 59 Briefly, male Sprague-Dawley
rats were anesthetized with sodium pentobarbital (35 mg/kg IV).
The left common carotid artery was exposed, and a 2F Fogarty
arterial embolectomy catheter was inserted into the
external carotid and passed down the common carotid to the aortic arch.
The balloon was expanded with 50 µL of saline and drawn back to the
bifurcation. This process was repeated three times. Bolus doses of
vehicle or test compounds were administered intravenously
at days -2, -1, and 0 relative to the angioplasty;
continuous infusion into the left jugular vein was initiated
immediately before angioplasty by use of preequilibrated Alzet osmotic
minipumps that were implanted subcutaneously in the back. Fourteen days
after angioplasty, animals under pentobarbital anesthesia
were euthanatized by placement of a catheter retrograde in the aorta
and flushing with saline at 100 mm Hg until the perfusate was
free of blood. This was followed by perfusion fixation with 10%
neutral buffered formalin.
Porcine Coronary Studies
Porcine angioplasty studies were conducted on juvenile male or
female Yorkshire pigs (
30 kg). Two days before angioplasty, animals
were anesthetized with 5% isoflurane (by mask inhalation) and
ketamine (300 mg IM), intubated, and maintained on 1.5% to
2.0% isoflurane and oxygen. An incision was made in the ventral neck
area, and an indwelling 10F dual-lumen catheter (Bard) was placed
in the jugular vein for withdrawal of blood samples and administration
of test compounds. The distal end of the catheter was exteriorized
between the scapulae on the dorsal thorax. Pigs were monitored by ECG
and pulse oximetry throughout the procedure. In the angioplasty
studies, administration of L-158,809 (1 mg/kg IV bolus followed by 1
mg·kg-1·d-1
constant IV infusion), L-163,082 (1.5 mg/kg IV bolus followed by 1.5
mg·kg-1·d-1
constant IV infusion), L-164,282 (1 mg/kg IV bolus followed by 1
mg·kg-1·d-1
constant IV infusion), or their respective vehicles was initiated
immediately after catheterization and withdrawal of
blood samples with EDTA anticoagulation for baseline plasma. Infusion
was driven by an external, programmable pump (CADD-PLUS, Pharmacia)
housed in a nylon vest worn by the pig. Animals were allowed to recover
in their home cages and were observed until stable.
For preliminary
experiments requiring blood pressure monitoring,
pigs were catheterized as described above. In addition, a vascular
access port (Access Technologies) was placed in the common carotid
artery and secured subcutaneously in the right lateral cervical area.
Arterial pressure was measured with DTX pressure transducer
systems (Viggo-Spectromed) connected to an ECG monitor (SpaceLabs).
Before administration of Ang II antagonists, changes in MAP
in response to bolus injections of Ang II (0.1 µg/kg IV) were
measured and used as an index for comparison with 
MAP measured in
the presence of antagonists.
Animals were medicated with 650 mg aspirin (24 hours before angioplasty) and 30 mg nifedipine (2 hours before). After 2 days of pretreatment with vehicle or test compound, animals were anesthetized as described above. The carotid artery was exposed and accessed through a catheter introduction sheath, and 10 000 U heparin (Upjohn) was administered intravenously. Under fluoroscopic imaging, the left anterior descending, left circumflex, and/or right coronary arteries were engaged by use of the appropriate coronary guide catheters. Isovue contrast medium (Squibb) was injected into each artery to determine the size of the PTCA balloon catheter needed. The balloon catheter, with a coiled tantalum wire stent wrapped around the balloon, was advanced to the desired location over a 0.014-in guide wire. The balloon was inflated to 8 atm for 15 seconds to expand the stent, resulting in a 1.2- to 1.4-fold ratio of balloon diameter to initial vessel diameter. The balloon was then deflated and withdrawn together with the guide wire, leaving the expanded stent in place. After administration of 1 g cefoxitin IV, animals were allowed to recover in their home cages and were maintained for 28 days after angioplasty with constant intravenous infusion of vehicle or test compound.
Jugular catheters were flushed aseptically with saline three times per week and were locked with 50% glucose/heparin to maintain patency. Blood samples were drawn weekly for estimates of plasma drug levels; plasma was stored at -70°C before assay. Ampicillin (500 mg PO) was given daily for the remainder of the study. On day 28 after angioplasty, animals were euthanatized with pentobarbital (60 mg/kg IV). Hearts were removed immediately and fixed with 10% neutral buffered formalin by pressure perfusion at 100 mm Hg.
Analysis of Neointimal Thickening
Five-micrometer cross
sections from
paraffin-embedded rat carotid arteries were stained with
hematoxylin-eosin. Neointimal cross-sectional areas
at three separate points along the injured segment were measured under
light microscopy by the JAVA morphometric system (Jandel). These
measurements were averaged to yield a single value for each vessel. For
the pig studies, arterial segments containing the expanded
intravascular stent were excised from formalin-fixed hearts and
were cut into 2-mm cross-sectional blocks. After careful removal of
stent wire fragments, the tissues were embedded in paraffin and
processed into 5-µm cross sections. Sections were stained with
hematoxylin-eosin and elastin–van Gieson. Sections from each
vessel were examined under light microscopy and were scored for degree
of injury (on a scale of 0 to 3 based on penetration of the stent wire
through the elastic laminae and medial smooth muscle layers) and
neointimal thickness (measured from the stent wire to the
adluminal aspect of the neointimal stenosis) as
described previously.52 Measurements made for each vessel
were averaged, such that each vessel gave rise to a single data
point.
Data Analysis
For the rat carotid studies, statistical
analysis was
performed on the natural logarithm of neointimal areas.
Consequently, the 90% CIs given are slightly asymmetrical about the
geometric means. Statistical comparisons between the treatment groups
were tested by the Wilcoxon rank sum test with a Bonferroni
adjustment to control the overall false-positive rate. For the
porcine studies, statistical analysis was performed on the
logarithm of neointimal thickness normalized for the degree
of injury. Specifically, after parallelism was checked for,
log(neointimal thickness) was regressed on injury score for
the injured vessels of antagonist-treated and control
pigs. Regression lines with equal slopes were fit to
log(neointimal thickness) for the treated and control pigs,
allowing the difference between treatment and control to be measured by
the difference in intercepts. A one-sided t test using
the jackknife procedure60 was used to formally test the
difference in intercepts between the antagonist-treated
and vehicle groups.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Rat Carotid Artery Angioplasty Studies
With the ultimate aim of comparing efficacy of Ang II receptor antagonists in vascular injury models of increasing complexity, our initial objective was to identify an effective antagonist and dosing strategy in the rat carotid artery. In this model, denudation of the vascular endothelium by balloon inflation and retraction provokes a reproducible formation of neointima, driven by migration and proliferation of medial SMCs.58 61 L-158,809, a potent, subtype-selective AT1 Ang II receptor antagonist, was chosen for angioplasty studies in this model. In vitro, L-158,809 exhibits high affinity for AT1 sites in rat aorta (IC50=0.3 nmol/L) and
30 000-fold
selectivity for AT1 versus AT2
sites.53 Similar binding potency was found for
AT1 receptors on rat carotid SMCs in culture (not shown).
In previous in vivo studies, bolus intravenous
administration of L-158,809 at 0.3 mg/kg to conscious rats blocked Ang
II–stimulated increases in systemic blood pressure (an
AT1-mediated response) by >80%; inhibition remained at
this level 6 hours after the bolus, declining to 60% after 24
hours.62
On the basis of these studies, the ability of
L-158,809 to inhibit
neointimal formation in the balloon-injured rat carotid
artery was examined in a series of animals that received bolus
intravenous doses of vehicle or L-158,809 (0.1, 0.3, or 1.0
mg/kg) on days -2, -1, and 0 relative to the balloon
procedure, followed by initiation of continuous intravenous
infusion (0.1, 0.3, or 1.0
mg·kg-1·d-1,
respectively) immediately after balloon injury. Treatment with 0.3 or
1.0
mg·kg-1·d-1
but not 0.1
mg·kg-1·d-1
L-158,809 produced statistically significant reductions in
neointimal areas measured 14 days after angioplasty (Table 1),
with maximal decreases of 37% and 35% occurring
with the 0.3- and 1.0-mg/kg doses, respectively. In a separate set of
animals receiving 0.1- or 1.0-mg/kg doses of L-158,809, blood was drawn
at intervals over the 16-day experimental course, and levels of
L-158,809 in plasma were determined. Intravenous infusion
of L-158,809 produced stable plasma levels ranging from 26 to 41 nmol/L
for the 0.1-mg/kg dose and 160 to 250 nmol/L for the 1.0-mg/kg dose
(Table 2). Since these plasma levels are far below
IC50 values estimated for L-158,809 at rat AT2
sites (10 µmol/L) and since the effective doses of L-158,809
produced prolonged inhibition of AT1-mediated pressor
responses,53 62 our results indicate that the
observed
inhibition of neointimal formation in the rat carotid
artery results from selective blockade of AT1 sites by
L-158,809. These findings are in concert with numerous other studies
using Ang II receptor antagonists in the rat carotid
angioplasty
model.10 11 43 44 45 46 47 48 49 50
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Pig Coronary Artery Angioplasty Studies
Activity of
L-158,809, L-163,082, and L-164,282 Against Pig Ang
II Receptors
The availability of nonpeptide antagonists with
distinct Ang II receptor selectivities allowed testing of the role of
Ang II in responses to vascular injury in a more complex system, the
porcine coronary artery. L-158,809, an
AT1-selective antagonist, was used to make a
direct comparison of the rat and pig vascular injury models. In
addition, L-163,082 (a balanced AT1/AT2
agent) and L-164,282 (an AT2-selective agent) were used to
test the possible contribution of Ang II acting through the
AT2 receptor in neointimal formation. Although
signaling via the AT2 site remains relatively poorly
characterized, evidence exists that AT2 may mediate an
antiproliferative response to Ang II.42 If such a
mechanism provides significant growth suppression in a
balloon-injured vessel, then selective blockade of AT2
sites might be expected to exacerbate neointimal
thickening.
The ability of L-158,809 and L-163,082 to block porcine
AT1
Ang II receptors in vitro was confirmed in binding studies using
monolayer cultures of porcine coronary artery SMCs (Fig 4A), in
which only the AT1 receptor subtype
is detectable. IC50 values for L-158,809 and L-163,082 were
0.5 and 3 nmol/L, respectively, measured in the presence of 0.1%
BSA; similar potencies were found toward rat AT1 receptors
(not shown). The lower apparent affinity of L-163,082 for
AT1 receptors in this cell-based assay compared with
membrane assays (IC50=0.3 nmol/L) reflects the presence of
BSA in the assay buffer. The relatively low affinity of the
AT2-selective compound L-164,282 toward the AT1
site also was evident in these experiments (IC50=0.6
µmol/L; Fig 4A).
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To verify the relative potencies of
L-158,809, L-163,082, and
L-164,282 toward the porcine AT2 receptor, competitive
binding studies were conducted using pig adrenal membranes as a source
of AT2 sites. Preliminary experiments revealed that 80% of
the specific [125I]Sar1Ile8Ang II
binding in pig adrenal membranes was attributable to AT1
sites (blocked by 1 µmol/L L-158,809 but not by the
AT2-selective ligand PD12331963 ), with the
remainder being inhibitable by PD123319. Therefore, 100 nmol/L
L-158,809 was added to subsequent binding incubations to block
AT1 sites. Residual specific binding, defined as
AT2 binding, was inhibited by either L-163,082 or L-164,282
with similar potencies (IC50 3 nmol/L in the presence of
BSA; Fig 4B). Thus, L-163,082 has the properties of a balanced
AT1/AT2 receptor ligand toward porcine
receptors, while L-164,282 shows an 200-fold selectivity for the
porcine AT2 site (0.6 µmol/L for AT1 versus 3
nmol/L for AT2).
In Vivo Activity of L-158,809,
L-163,082, and
L-164,282
The pressor effect of Ang II is a well-defined
AT1-mediated physiological response,
and blockade of this response would be expected to reflect circulating
antagonist levels sufficient also to block the
growth-promoting effects of Ang II in vascular tissue. Therefore,
to identify appropriate dosing levels of the AT1-reactive
antagonists in the pig, we determined rates of infusion
sufficient to block increases in systemic blood pressure stimulated by
exogenous Ang II. Infusion of L-158,809 at 0.5
mg·kg-1·d-1
produced mean plasma levels of compound of 25 nmol/L and 75%
inhibition of Ang II pressor response; infusion at 1.0
mg·kg-1·d-1
gave rise to 150 nmol/L plasma concentration and >85% pressor
blockade (Fig 5A). In two normotensive animals tested,
infusion of L-158,809 at 1.0
mg·kg-1·d-1
also was associated with a 16% decrease (after 26 days) and a 36%
decrease (after 12 days) in baseline MAP. The 1.0
mg·kg-1·d-1
infusion rate was chosen for the angioplasty studies to achieve
effective blockade of AT1 receptors while producing plasma
levels far below the IC50 for blockade of AT2
receptors (>10 µmol/L). Higher rates of infusion were considered
inappropriate owing to possible additional effects on baseline MAP or
interaction with AT2 sites. In three animals that received
a 1-mg/kg bolus of L-158,809 followed by 48-hour infusion at 1
mg·kg-1·d-1,
the Ang II pressor dose-response curve was shifted rightward by
100-fold (data not shown). The magnitude of this shift exceeds that
reported by Kauffman et al44 (55-fold) to be necessary for
the inhibition by losartan of rat carotid
neointimal formation.
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Similar preliminary studies were performed to
establish appropriate
L-163,082 dosing (Fig 5B). Infusion was initiated at 1.5
mg·kg-1·d-1,
and Ang II responses and plasma drug levels were determined over a
period of days. Plasma levels of L-163,082 150 nmol/L were associated
with 80% blockade of Ang II pressor responses. There was no
consistent effect of L-163,082 infusion at this dose on
baseline MAP over a period of 28 days in a single animal tested. Since
L-163,082 was observed to be equipotent at porcine AT1 and
AT2 sites (Fig 4), the demonstrated
AT1-blocking dose in the pig was assumed to be sufficient
also to occupy AT2 sites.
Since there is no well-accepted
physiological
response mediated by AT2 stimulation that can be monitored
in the short term, establishing appropriate dosing levels with
AT2-selective agents such as L-164,282 is
problematic. To address the dosing issue, a series of
intravenous bolus doses of L-164,282 (1 to 5 mg/kg) was
administered. At various times after each bolus, blood was drawn for
estimation of plasma L-164,282 concentrations, and maximal increases in
MAP after an Ang II challenge were measured. In these experiments,
plasma levels of L-164,282 of <5 µmol/L were associated with <20%
inhibition of the pressor response to Ang II, indicating little or no
blockade of AT1 receptors (Fig 6). In
contrast, a 1-mg/kg bolus of L-158,809, an AT1-selective
antagonist, totally abolished pressor responses to Ang II
in these same experiments (not shown). An intravenous bolus
of L-164,282 (1 mg/kg) followed by continuous infusion at 1
mg·kg-1·d-1
yielded plasma levels of 140 to 950 nmol/L measured over a period of 6
days. These plasma levels correspond to concentrations 35 to 240
times the observed IC50 for L-164,282 versus the porcine
AT2 receptor (3 nmol/L) but were not associated with
appreciable blockade of an AT1-mediated pressor response in
the pig. Therefore, the
1-mg·kg-1·d-1
dose was chosen for the angioplasty studies.
|
Plasma Levels
of Test Compounds in Study Pigs
In the group of animals treated with
L-158,809, mean plasma levels
of compound were 130 nmol/L on the day of angioplasty and 370 nmol/L on
the day they were killed (Table 3), ie, above the levels
determined independently to be sufficient for AT1 blockade
(Fig 5). Similarly, mean plasma levels of L-163,082 in study
pigs were
above those required for pressor blockade, ranging from 260 nmol/L on
the day of injury to 1020 nmol/L after 3 weeks. Mean plasma levels of
L-164,282 were 24 nmol/L on the day of angioplasty and ranged from 260
to 720 nmol/L over the 28-day postangioplasty course, ie, 8- to
240-fold above the IC50 for L-164,282 versus the porcine
AT2 receptor but well below levels associated with evidence
of AT1 blockade.
|
Effects of L-158,809,
L-163,082, or L-164,282 on
Neointimal Thickness
Data for neointimal thickness and injury scores
from
vehicle and compound-treated animals are shown in Fig 7. Mean
injury scores did not differ between vehicle-
and antagonist-treated groups in any of the three
studies (Table 4). For the L-158,809 studies, the
estimated ratio of mean neointimal thicknesses for treated
versus vehicle groups was 0.872 (P=.12). In the L-163,082
studies, this ratio was 1.045 (P=.75) and in the L-164,282
studies, 0.997 (P=.48). Thus, there was no evidence that
treatment with either L-158,809, L-163,082, or L-164,282 in these
experiments had a major effect on neointimal thickness
after angioplasty in the pig coronary artery.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
The establishment of appropriate animal models for the study of restenosis remains a major challenge.64 65 The ballooned rat carotid artery model of vascular injury has been widely used both to study the basic pathophysiology of responses to balloon injury and to test potential therapeutic agents. In this model, formation of neointima can be rapidly induced and reliably measured. However, the rat model has been disappointing as a predictor of efficacy in other species. More complex models of vascular restenosis have been developed with the aim of generating a body of experimental data with greater predictive power for efficacy in humans. Toward this end, porcine coronary vascular injury models, systems that appear to approximate human pathophysiology more closely than does the rat model,51 66 67 68 are being used with increasing frequency. The pig has a coronary size and vascular distribution similar to those of humans and develops a neointima that more closely resembles human restenotic lesions grossly and microscopically. Degrees of injury and neointimal response are readily quantifiable in this model.52 While this model awaits validation with respect to predicting human efficacy, it is important to note that a limited number of agents, including the factor Xa inhibitor tick anticoagulant peptide69 and the thrombin inhibitor hirudin70 have been effective at reducing neointimal thickening in stented pig coronary studies.
A prominent example of the discrepancy between results of vascular injury studies in rats and larger animals is the failure of ACE inhibitors to improve the long-term outcome of angioplasty in humans22 23 24 or to inhibit neointimal thickening in porcine models,18 19 20 despite numerous reports of successful neointimal inhibition in the rat.7 8 9 10 11 The simplest interpretation of these contrasting results is that Ang II derived from ACE-dependent pathways is a major mediator of intimal hyperplasia in rats but not in humans. Sensitivity to ACE inhibitors will be dictated in part by the relative contributions of ACE-dependent and non–ACE-dependent mechanisms of Ang II biosynthesis in the two species. The importance of this factor is suggested by the detection of chymase activity capable of significant Ang II generation in human but not rat arteries.27 28 In addition, doses of ACE inhibitors used in human restenosis studies, although effectively antihypertensive, were far below those associated with inhibition of neointimal formation in rat studies.7 8 9 10 11 22 23 24 Thus, even if ACE were the sole generator of Ang II in the human heart, the possibility exists that the supra-antihypertensive doses of ACE inhibitors needed to effectively inhibit tissue ACE15 may lie outside the tolerable range for humans.
The availability of nonpeptide antagonists of Ang II receptors has offered the opportunity to test pathophysiological roles of Ang II independent of its means of generation. In the present studies, nonpeptide Ang II receptor ligands were evaluated for their ability to affect intimal hyperplasia in a porcine coronary artery model of vascular restenosis. Administration of the AT1-selective antagonist L-158,809, while capable of inhibiting neointimal area in rat carotid studies by 37%, did not have statistically significant effects on neointimal thickening after intravascular stent placement in pig coronary arteries. Similarly, L-163,082 (a balanced AT1/AT2 ligand) and L-164,282 (an AT2-selective agent) did not reduce neointimal thickening. Each compound was administered by constant intravenous infusion over the entire 30-day study time course (2 days pre-PTCA through 28 days post-PTCA), chronically maintaining circulating levels sufficient to block the targeted receptor subtype(s).
These results suggest that Ang II, acting through the AT1 receptor, is not a major mediator of coronary intimal hyperplasia in the injured porcine coronary artery. In addition, our results suggest that the AT2 site does not play a major growth-suppressing role in this model, since neither a selective AT2 ligand nor a balanced AT2 ligand produced significant exacerbation of neointimal thickening. It should be noted, however, that since the nature of signaling through the AT2 receptor remains obscure, it is not possible to fully predict the consequences of (1) Ang II signaling through AT2 while AT1 is blocked (eg, in the presence of L-158,809), (2) AT1 signaling while AT2 is blocked, or (3) the potential activation of the AT2 receptor by nonpeptide AT2 ligands. Therefore, all results using site-selective Ang II antagonists in these and other studies will require reevaluation as more is learned about AT2 signaling. Our results further suggest that previous findings that ACE inhibitors are ineffective at reducing neointimal thickening in the pig18 19 20 do not stem from a failure of ACE inhibitors to inhibit Ang II generation but rather from a relatively minor contribution of Ang II to neointimal thickening in this species. This conclusion also is consistent with the recent finding that all detectable Ang II generation from Ang I in the isolated pig coronary artery, even that occurring in the media or adventitia, could be blocked by ACE inhibitors.71
The present results using site-selective Ang II receptor antagonists, together with previous porcine studies using ACE inhibitors,18 19 20 demonstrate a marked dissociation between results gained with rat and porcine models: whereas Ang II appears to play a major role in neointimal formation in the rat carotid artery, a role of similar magnitude cannot be inferred in the pig coronary artery. The reasons for this discrepancy are unclear, but it may derive from a combination of vessel and species differences. The nature of the injuries imparted in the two models is clearly different: balloon dilatation of the relatively elastic rat carotid artery is characterized by endothelial denudation, minimal thrombus formation, and medial damage by overstretching, whereas medial and even adventitial dissection and robust thrombus formation are common after pig coronary angioplasty. These distinct injuries thus may provoke patterns of response that differ in their sensitivity to specific inhibitors. Ang II may serve as a major proliferative or migratory stimulus in the injured rat carotid but not in the pig coronary artery. Although Ang II has been characterized as, at best, a weak mitogen toward rat (reviewed in References 4 through 6) or human vascular SMCs in vitro,72 73 Ang II treatment does potentiate neointimal formation in the balloon-injured rat carotid artery.12 Thus, the precise activities of Ang II in vivo are difficult to predict. Ang II potentially may interact with other growth-regulating substances with either stimulatory (eg, epidermal, platelet-derived, or insulin-like growth factors) or inhibitory (eg, transforming growth factor-ß) properties.74 75
Alternatively, distinct interspecies effects of Ang II on neointimal development may stem from differential modulation of cell–extracellular matrix interactions. Migration of SMCs from the medial to intimal compartments has been estimated to rival cell proliferation as a factor contributing to neointimal thickening in the rat,61 76 and an ACE inhibitor was reported to reduce vascular SMC migration in vitro.77 The ineffectiveness of ACE inhibitors or Ang II receptor antagonists in reducing intimal hyperplasia in the porcine coronary artery, then, may indicate that factors other than Ang II are the major mediators of migration or could reflect a relative unimportance of cell migration in lesion development.
The value of any animal model of human disease can be proved only when clearly effective human treatments exist for comparative purposes. Until such treatments are identified for vascular restenosis, indirect means must be used to evaluate existing experimental models. Although the value of the rat carotid injury model has been questioned on the basis of its poor predictive record for human efficacy, it seems likely that rat studies may be well suited for identifying inhibitors of certain mechanisms, such as cell migration or extracellular matrix elaboration, that contribute to restenosis in humans. Other species- or target vessel–dependent factors remain to be fully evaluated, including the relative contributions of platelet-rich thrombi or medial/adventitial dissection, which frequently occur in both human and pig coronary arteries after angioplasty,51 52 60 to the pathogenesis of the final lesion. These similarities notwithstanding, our results do not exclude a possible role for Ang II in the vascular response to angioplasty in humans. Thus, it remains to be determined whether the absence of effect of Ang II receptor antagonists in porcine coronary studies would be predictive of lack of efficacy in human restenosis trials.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
We express our appreciation to Dr W. John Powell for his constructive critiques of the model, protocol, and data; to Dr William Grossman for his helpful suggestions; to Drs Peter Siegl and Salah Kivlighn for furnishing information on the receptor antagonist compounds; and to Drs Hilton Klein, Tom Nolan, and Joseph Lynch for their enthusiasm and for supplying technical support from their respective groups.
Received September 6, 1995; revision received October 19, 1995; accepted October 30, 1995.
|
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S. Hafizi, X. Wang, A. H. Chester, M. H. Yacoub, and C. G. Proud ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1232 - H1238. [Abstract] [Full Text] [PDF]
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W. R. Huckle, M. D. Drag, W. R. Acker, M. Powers, R. C. McFall, D. J. Holder, T. F. Walsh, R. S. Schwartz, W. J. Greenlee, and R. G. Johnson Jr Effects of L-749,329, an ETA/ETB Endothelin Receptor Antagonist, in a Porcine Coronary Artery Injury Model of Vascular Restenosis Circulation, April 10, 2001; 103(14): 1899 - 1905. [Abstract] [Full Text] [PDF]
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J. Ibrahim, A. D. Hughes, and P. S. Sever Action of Angiotensin II on DNA Synthesis by Human Saphenous Vein in Organ Culture Hypertension, November 1, 2000; 36(5): 917 - 921. [Abstract] [Full Text] [PDF]
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S. Patel, Y. Shi, R. Niculescu, E. H. Chung, J. L. Martin, and A. Zalewski Characteristics of Coronary Smooth Muscle Cells and Adventitial Fibroblasts Circulation, February 8, 2000; 101(5): 524 - 532. [Abstract] [Full Text] [PDF]
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D. P. Wilson, L. Saward, P. Zahradka, and P. Kee Cheung Angiotensin II receptor antagonists prevent neointimal proliferation in a porcine coronary artery organ culture model Cardiovasc Res, June 1, 1999; 42(3): 761 - 772. [Abstract] [Full Text] [PDF]
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K. Harada, I. Komuro, T. Sugaya, K. Murakami, and Y. Yazaki Vascular Injury Causes Neointimal Formation in Angiotensin II Type 1a Receptor Knockout Mice Circ. Res., February 5, 1999; 84(2): 179 - 185. [Abstract] [Full Text] [PDF]
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A. Lafont and D. Faxon Why do animal models of post-angioplasty restenosis sometimes poorly predict the outcome of clinical trials? Cardiovasc Res, July 1, 1998; 39(1): 50 - 59. [Full Text] [PDF]
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R. E. Pratt and V. J. Dzau Pharmacological Strategies to Prevent Restenosis : Lessons Learned From Blockade of the Renin-Angiotensin System Circulation, March 1, 1996; 93(5): 848 - 852. [Full Text]
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