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Circulation. 1996;93:1009-1019

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(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

William R. Huckle, PhD; Marlene D. Drag, DVM; Wayne R. Acker, MS; Michele Powers, BS; Rosemary C. McFall, BS; Daniel J. Holder, PhD; Tsuneo Fujita, BS; Inez I. Stabilito, BS; Dooseop Kim, PhD; Debra L. Ondeyka, BS; Nathan B. Mantlo, PhD; Raymond S.L. Chang, PhD; Christopher F. Reilly, PhD; Robert S. Schwartz, MD; William J. Greenlee, PhD; Robert G. Johnson Jr, MD, PhD

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.


*    Abstract
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 *Abstract
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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


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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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 1Down). 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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  		Figure 1. Pathways of Ang II generation and activity.

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 1Up),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.


*    Methods
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 *Methods
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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 2Down), 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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  		Figure 2. Structures of L-158,809, L-163,082, and L-164,282.

Structural analogues of L-163,082 (Fig 2Up) 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 3Down. Nitration of 2-amino-4-methyl-3-nitropyridine ([1] in Fig 3Down) provided 2-amino-4-methyl-3,5-dinitropyridine ([2] in Fig 3Down). 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 2Up). 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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  		Figure 3. Synthesis of L-163,082. a, HNO3 (1 equivalent), H2SO4; 0°C to room temperature, 24 hours; b, H2 (1 atm), Raney nickel (5%), 1:1 THF-MeOH; c, filtered (under N2) into 3 equivalents of concentrated HCl, then concentrated under vacuum; d, PrCO2H (3 equivalents), polyphosphoric acid, 80°C, 8 hours; e, CsCO3, 4-bromomethyl-3-fluoro-2'-tert-butylamino-sulfonyl[1,1']biphenyl, dimethylformamide (DMF), room temperature, 3 to 8 hours; f, trifluoroacetic acid, 24 hours, room temperature; g, 3:1 concentrated aqueous HCl/MeOH, 60°C, 12 hours; h, PhCOCl (1 equivalent), triethylamine (2 equivalents), 5:1 THF-DMF, -20°C to 0°C; i, n-BuOCOCl (3 equivalents), dimethylamino pyridine (3 equivalents), pyridine, room temperature, 2 to 12 hours.

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 ({approx}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 {Delta}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.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
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 {approx}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 1Down), 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 2Down). 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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  		Table 1. Inhibition of Neointimal Formation in the Rat Carotid Artery by L-158,809


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  		Table 2. Plasma Levels of L-158,809 After Intravenous Bolus and Infusion in Rats

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 4ADown), in which only the AT1 receptor subtype is detectable. IC50 values for L-158,809 and L-163,082 were {approx}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 4ADown).



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  		Figure 4. Inhibition of Ang II radioligand binding by L-158,809, L-163,082, or L-164,282. A, Ang II receptor binding in porcine coronary artery SMCs (VSMC) was conducted as described in "Methods." Data (mean±SD, n=3) are expressed as percent specific AT1 binding (total binding minus that occurring in the presence of 1 µmol/L nonlabeled Sar1Ile8Ang II). B, Ang II receptor binding in porcine adrenal membranes was conducted as described in "Methods." Preliminary experiments revealed that {approx}20% of the specific [125I]Sar1Ile8Ang II binding was attributable to AT2 sites (blocked by 1 µmol/L PD123319 but not by 1 µmol/L L-158,809). For the results shown, incubations contained 100 nmol/L L-158,809 to block AT1 sites. Data (mean±SD, n=3) are expressed as percent specific AT2 binding (binding in the presence of L-158,809 minus that in the presence of 1 µmol/L nonlabeled Sar1Ile8Ang II).

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 {approx}3 nmol/L in the presence of BSA; Fig 4BUp). Thus, L-163,082 has the properties of a balanced AT1/AT2 receptor ligand toward porcine receptors, while L-164,282 shows an {approx}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 {approx}25 nmol/L and {approx}75% inhibition of Ang II pressor response; infusion at 1.0 mg·kg-1·d-1 gave rise to {approx}150 nmol/L plasma concentration and >85% pressor blockade (Fig 5ADown). 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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  		Figure 5. Inhibition of Ang II pressor response during infusion of L-158,809 or L-163,082. Top, Pigs were infused intravenously with L-158,809 at 0.1, 0.5, or 1.0 mg·kg-1·d-1 as indicated. Immediately before Ang II challenge, blood was drawn for estimation of plasma L-158,809 concentration. Maximum changes in MAP after Ang II challenge (0.1 µg/kg IV) then were measured. Pressor responses in the presence of L-158,809 are expressed as percent inhibition of the maximum {Delta}MAP recorded before L-158,809 administration. Bottom, Percent inhibition of Ang II–stimulated {Delta}MAP and L-163,082 plasma concentrations during infusion of L-163,082 (1.0 mg·kg-1·d-1) were determined as described for A.

Similar preliminary studies were performed to establish appropriate L-163,082 dosing (Fig 5BUp). 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 4Up), 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 6Down). 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 {approx}35 to 240 times the observed IC50 for L-164,282 versus the porcine AT2 receptor ({approx}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.



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  		Figure 6. Status of Ang II pressor response during infusion of L-164,282 in the pig. A series of bolus doses of L-164,282 (1 to 5 mg/kg IV) 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, as described in "Methods."

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 3Down), ie, above the levels determined independently to be sufficient for AT1 blockade (Fig 5Up). 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.


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  		Table 3. Plasma Levels of L-158,809, L-163,082, and L-164,282 in Study Pigs

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 7Down. Mean injury scores did not differ between vehicle- and antagonist-treated groups in any of the three studies (Table 4Down). 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.



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  		Figure 7. Plots of neointimal thickness vs injury scores after PTCA in the pig. The natural logarithm of mean neointimal thickness (in millimeters) is plotted versus mean injury score for each injured vessel recovered from animals treated with vehicle or the Ang II receptor ligands L-158,809 (top), L-163,082 (middle), or L-164,282 (bottom). Regression lines determined by use of the jackknife analysis are shown for vehicle (solid curve) and treated (broken curve) groups.


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  		Table 4. Mean Injury Scores and Neointimal Thickness


*    Discussion
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*Discussion
down arrowReferences
 
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
 
ACE = angiotensin-converting enzyme
Ang II = angiotensin II
MAP = mean arterial pressure
PTCA = percutaneous transluminal coronary angioplasty
SMC = smooth muscle cell
WBH = Waymouth's medium containing 0.1% BSA (fraction V, Sigma) and 20 mmol/L HEPES, pH 7.3


*    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.


*    References
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 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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