© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Altered endothelin-1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252
aDepartment of Chemical Pathology and Human Metabolism, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK
bDepartment of Cardiovascular Pharmacology, Knoll AG, P.O. Box 210805, D-67008 Ludwigshafen, Germany
cDepartment of Biostatistics and Data Management, Knoll AG, P.O. Box 210805, D-67008 Ludwigshafen, Germany
* Corresponding author. Tel.: +49-621-5892559; fax: +49-621-5891405 klaus.muenter{at}knoll-ag.de
Received 12 August 1998; accepted 1 February 1999
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Abstract |
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Objective: Since raised levels of endothelin-1 (ET-1) have been detected in the human coronary sinus following percutaneous transluminal angioplasty (PTCA) we investigated the role of ET-1 in the etiology of vascular restenosis. Methods: Balloon angioplasty of coronary arteries was performed in pigs and the animals were treated with placebo or the endothelin (ETA) receptor antagonist LU 135252 (30 mg/kg/day). After 4 weeks vascular stenosis and the distribution of endothelin and its receptors was evaluated. Results: The pronounced neointima formation in the control group (neointima:media ratio=0.87±0.36) was significantly reduced by LU 135252 (0.43±0.30, P<0.001). Angioplasty caused a significant increase in medial ETA (approximately 275%, P<0.026) and ETB (approximately 250%, P<0.001) binding to injured, compared with non-injured segments, an effect that was also reduced by LU 135252 (ETA=11.5% increase; ETB=14% increase). The neointima of control animals exhibited ET-1 like immunoreactivity as well as ETA and ETB binding sites. Conclusion: These data indicate that endothelin is locally-released from endothelial and vascular smooth muscle cells following angioplasty which binds to ETA and ETB receptor sites in the neointima and media. Since administration of the ETA antagonist LU 135252 markedly reduces neointima formation and medial ET binding, we conclude that vascular smooth muscle cell proliferation and subsequent neointima formation is mediated predominantly via ETA receptors. These data underscore the therapeutic potential of ETA antagonists in reducing the degree of restenosis following vascular injury.
KEYWORDS Experimental; Vasculature; Pharmacology angioplasty; Coronary arteries; Endothelin receptors; Restenosis; Histology
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The long-term efficacy of percutaneous transluminal coronary angioplasty (PTCA) is limited by the high incidence of vascular stenosis following this procedure. The damaging effect of PTCA on vascular endothelial cells, and underlying tissue, is known to play a significant role in neointimal formation, via the release of a variety of neurohumoral compounds. Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide [1] also possesses mitogenic activity on various cells types, including vascular smooth muscle cells [2]. Levels of ET-1 are elevated in the human coronary sinus following PTCA, suggesting that this peptide plays a pathological role in vascular restenosis [3].
There is evidence that exogenous administration of ET-1 causes a dose-dependent augmentation of angioplasty-induced neointimal formation of the rat carotid artery [4]. Recently it has also been shown that ET receptor antagonists reduce neointimal formation in this animal model, suggesting that endogenous ET-1 is involved in angioplasty-induced lesion formation in the rat [5,6]. These studies have used a combination of morphometric analysis and vascular smooth muscle cell (VSMC) culture/[3H]thymidine incorporation assay to study the potential proliferative role of ET-1 in neointimal formation in the rat carotid artery following balloon angioplasty. Similar results have recently been described following oral administration of the ETA receptor antagonist LU 135252 [7]. Here we have performed PTCA on porcine epicardial coronary arteries and examined the effect of the ETA receptor antagonist, LU135252, on subsequent neointima formation in these animals. Vascular stenosis was assessed morphometrically while the localization of ET-1 and its receptors (ETA/ETB) was studied using a combination of immunohistochemistry and in vitro autoradiography.
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2 Methods |
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Twelve male domestic pigs, 8–9 weeks old weighing 22–29 kg were used. The animals were acclimatized for at least 1 week prior to the experiment. Animal care and husbandry conformed with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985), were in compliance with EC directive 86/609 and the study was approved by the appropriate local authority.
2.1 Balloon angioplasty
All animals were given 2 mg/kg stresnil (Azaperon®) i.m., followed by 4 mg/kg metomidat (Hypnodil®, Janssen) i.v. Animals were intubated and ventilated with 75% N2O and 25% O2. A standard PTCA guide catheter (Judkin left, powerbase 8F, ACS:mandrin) was then advanced via the aortic root into the left anterior descending coronary artery (LAD) under X-ray guidance. The balloon catheter (RX Elipse 0.014, ACS) was positioned in the middle portion of the vessel using a 0.014'' guide wire (Galeo, Biotronic, Berlin). After confirming its position under X-ray control the balloon catheter was inflated twice at 10 atm for 30 s. The balloon to artery ratio was 3 mm to 1.6–2 mm. After deflating the balloon the catheter was withdrawn and an additional angiogram made to verify the lesion. Additionally, the degree of vessel injury at the angioplasty site was shown to be the same for both groups by histological examination post mortem.
2.2 Drug administration
Before PTCA the pigs were randomised into two groups, one receiving 30 mg/kg/day LU 135252 orally (mixed into mashed potatoes) or controls. Immediately prior to balloon inflation the animals in the respective groups received either 3 mg/kg LU 135252 or 0.9% NaCl intravenously. Oral treatment was started on the first day after PTCA and continued for 4 weeks. LU 135252 (+)-(S)-(2-(4,6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenyl-propionic acid) was synthesised at BASF, Ludwigshafen. LU 135252 is a selective ETA receptor antagonist (Ki=1.4 nM) with more than 100-fold selectivity over ETB receptors (Ki=184 nM). No binding to other receptors was found up to 10 µM. The dose of 30 mg/kg/day was chosen according to Ref. [7] where a selective blockade of ETA receptors was achieved. The cardiovascular effects of LU 135252 were investigated in pigs after administration of 10 mg/kg i.v. This dose had no effect on blood pressure (MK, data on file). As the peak plasma concentration reached after 10 mg/kg i.v. was higher than after 30 mg/kg p.o. given with the feed it was assumed that no haemodynamic effects would occur in this study.
2.3 Tissue collection
Twenty eight days after balloon angioplasty the animals were anaesthetised, the chest opened and the heart removed. The dilated coronary artery segments, as determined from X-ray pictures of exact balloon position and the adjacent non-injured vessel segments, were then carefully dissected from the epicardial surface and transferred into phosphate-buffered saline and tissue tek injected into the lumen of the artery. After freezing, the artery was divided into three injured and one proximal non-injured transverse segments (5 mm each) and stored at –70°C until use. Additionally, one non-injured segment of the same artery was removed (see Section 2.5).
2.4 Morphometry
Three 10-µm transverse sections from each injured vessel segment were cut in a cryostat, thaw-mounted onto microscope slides and stained with haematoxylin and eosin. Non-injured control segments were obtained from the same animals and the same vessel proximal to the balloon injury. In each section, the cross sectional area of lumen, media and neointima and total vessel was determined by a person who was blinded with regard to the treatment groups using digital morphometry (cardio 200). Ratios of neointima to media area and to total vessel area were calculated.
2.5 In vitro autoradiography
ET-1 receptors were identified as described previously [8]. Briefly, 10-µm serial transverse sections of injured and non-injured segments of the LAD were cut in a cryostat at approximately –20°C and thaw-mounted onto gelatinised microscope slides. Endogenous peptide levels were reduced by preincubating sections in 50 mmol/l Tris–HCl buffer, pH 7.4, for 15 min at 22°C. Slide-mounted tissue was then incubated in Tris–HCl buffer (plus 5mmol/L MgCl2, 0.2% bovine serum albumen and 100 kIU/ml aprotinin) containing 100 to 150 pmol/l [125I]ET-1 (specific activity 2000 Ci/mmol/l, Amersham, Buckinghamshire, UK) for 120 min at 22°C. ETA binding sites were identified by incubating sections in [125I]-PD151242 (=(N-[(hexahydro-1-azepinyl)carbonyl])L-Leu(1-Me)D-Trp-D-Tyr, [9,10]) and ETB binding sites using [125I]-BQ3020 (=N-Acetyl-[Ala11,15]-endothelin-1 (6-21), [10,11]) 100–150 pmol/l, specific activity of both compounds 2000 Ci/mmol/l; Amersham). The degree of non-specific binding for each radioligand was established by incubating alternate slides in the presence of 1 µmol/l unlabelled ET-1 (Bachem Fine Chemicals, Basel, Switzerland). After incubation tissue was washed (two times 10 min) in Tris–HCl buffer (4°C), dipped in ice-cold glass-distilled water, and dried in a stream of cold air.
Autoradiographs were generated by exposing incubated tissue to Hyperfilm 3H (Amersham International, Buckinghamshire, UK) in X-ray cassettes for up to 4 days at 4°C. After this period films were processed according to the manufacturer’s instructions. Receptor binding was assessed densitometrically on a Kontron VIDAS imaging system (Kontron Ltd, Thame, Oxfordshire, UK) and binding calculated from curves generated by [125I] microscales (Amersham) that were co-exposed with slide-mounted tissue. Binding was localised at the cellular level by dipping incubated sections in molten nuclear emulsion (Hypercoat LM-1, Amersham) and exposing for up to 8 days at 4°C in light-proof boxes. Emulsion was processed and underlying tissue stained with Mayer’s haematoxylin and eosin for histological examination [12].
2.6 Immunohistochemistry
Immunohistochemistry was performed on selected sections of coronary artery using the Vector avidin–biotin complex method (ABC) (Vector Laboratories, Peterborough, UK). Briefly, sections were fixed in acetone, rinsed, blocked in 20% normal goat serum in Tris-buffered saline and incubated for 20 h at 10°C in monoclonal anti-ET-1 [13,14] (generously provided by Dr. A.C.M. Ong) (diluted 1:500). Tissue was then processed with biotinylated goat anti-mouse IgG and Vector’s ABC kit, developed with either 3,3'-diaminobenzidine or Vector red and counterstained with Mayer’s haematoxylin.
Smooth muscle cells were identified using an anti-alpha-smooth-muscle actin antibody (Dako Laboratories, High Wycombe, Buckinghamshire, UK). Controls were established using an irrelevant antibody and no primary antibody.
VSMC nuclei counts have been carried out on five fields per vessel section (within a grid measuring 100x50 µm) on 4–6 sections from injured and non-injured segments from five animals (at x200 magnification). The mean±S.E.M. of 20–30 measurements per [injured/non-injured] vessel was calculated from n=five animals: >250 counts).
2.7 Statistical analysis
Mean section values of lumen, media, neointima, total vessel area as well as the indexes neointima/media and neointima/total area of both groups were averaged and described with summary statistics. Observed data of the different tissue areas were analyzed with a multiway analysis of variance (factors drug (1=control, 2=LU 135252), animal (1–5 in the control group and 5–12 in the LU 135252 group), location of sections (1–3), repetition (1–3)), with the factors animal, location, repetition nested within the drug factor. All statistical analysis was performed with SAS version 6.12 (SAS Institute Inc, Cary, NC).
Calculated receptor densities in injured and non-injured medial tissue were averaged and described with a general linear model (ANOVA) using the same SAS software. Differences were considered significant at the level P<0.05.
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3 Results |
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In control animals histological examination revealed neointima formation within segments of LAD one month following PTCA. The neointima was evident as a pronounced eccentric layer, composed largely of smooth muscle cells (positive anti-alpha-actin immunoreactivity), underlying a ruptured internal elastic lamina (Fig. 1), often at more than one site. Table 1 shows the observed section means and summary statistics for the different areas, obtained over all animals and sections included in the control and LU 135252 group, Table 2 the data of non-injured LAD segments, and Table 3 the ANOVA results. The total area of non-injured segments was slightly larger as these segments were located proximally to the injury site. The proliferative response (expressed as neointimal area) in injured segments was significantly reduced in animals receiving LU 135252 (Table 1 and Figs. 1 and 2
). Although the medial area was slightly larger in LU 135252 treated pigs, the neointima/media ratio was still reduced compared to the control group (0.43±0.30 vs. 0.87±0.36, P<0.001). The observed differences in luminal area between treatment groups were statistical significant. In order to detect any possible influence on vascular remodeling we further investigated the differences in total vessel area and the ratio neointima to total vessel area in injured segments (Table 1). No significant difference with respect to the total area between control and treatment group was detected (Table 1). The index neointima/total area was statistically significant smaller in LU 135252-treated pigs compared to placebo-treated pigs indicating a significant positive effect of ETA receptor blockade after PTCA (Tables 1 and 3). The observed differences between the pigs were significant as well as the observed differences between section areas. No statistical significant differences between the three replicate measurements made on each segment of all areas (Tables 1 and 3) were observed.
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The dense [125I]-ET-1 binding to all vessel sections (Fig. 3) that was markedly reduced where tissue was incubated in the presence of unlabelled ET-1 (non-specific binding [NSB] of [125I]-ET-1 <6% of total binding; [125I]-PD151242) <11% NSB; [125I]-BQ3020 <18% NSB). This binding was mainly associated with the tunica media, and predominantly to ETA ([125I]-PD151242) binding sites (Fig. 4). There was also ETB ([125I]-BQ3020) binding to the tunica media, with the ETA/ETB ratio being approximately 1.6:1 (Figs. 4 and 7
). Medial binding to injured compared to non-injured segments of control animals (n=6) was significantly elevated (specific ETA binding, injured=33.2±7.9 vs. non-injured=12.1±1.5 dpmx103/mm2, P<0.026; ETB, injured=19.4±2.4 vs. non-injured=7.8±0.8 dpmx103/mm2, P<0.001; Fig. 5) and this injury-induced effect was reduced (to control values) in animals receiving LU 135252 (Fig. 5). There was no significant effect of drug-treatment on ETA/ETB binding to control (non-injured) vessel segments. VSMC number per grid area (100x50 µm) in non-injured section was 29.7±1.5 vs. 28.6±2.9 in injured segments. Since no significant difference in VSMC density between injured and non-injured vessels was detected we conclude that increased binding (in injured vessels) is due to increased binding per cell rather than being secondary to an increase in cell density. On the film autoradiographs (low resolution) dense binding to the tunica media of injured segments was evident, with lower binding to regions of neointima formation (see Figs. 3, 4, 6 and 7b). The neointimal binding was predominantly to ETA sites (Figs. 4 and 7b; specific [125I]-PD151242 binding=9.5±2.4x103 dpm/mm2, compared to 5.5±0.5x103 dpm/mm2 specific [125I]-BQ3020 binding; mean±S.E.M. of n=5 pigs).
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Autoradiographs, obtained by coating slide-mounted sections with nuclear emulsion (high-resolution), confirmed that there was dense binding of all radioligands to the tunica media (Fig. 6) and that, at this region, ETA binding was stronger than ETB binding (Figs. 7a,b). Histology of non-injured segments of LAD confirmed that luminal endothelial cells were intact and in close apposition to an undamaged internal elastic lamina (Fig. 7a). In these segments binding was essentially uniform throughout the vessel wall. All injured segments of control animals showed the formation of neointima underlying a ruptured internal elastic lamina (Figs. 6 and 7b
). The damage to the internal elastic lamina was often to several sites and, on occasions, extended to the external elastic lamina.
The immunohistochemical studies confirmed that the neointima was composed mainly of smooth muscle cells (positive anti-alpha smooth muscle actin reactivity) and that these proliferating cells exhibited ETA and ETB binding (Figs. 4 and 7b) as well as positive ET-1-like immunoreactivity (Fig. 8).
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4 Discussion |
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The vasoconstrictor peptide ET-1 also has a potent mitogenic action on vascular smooth muscle cells [15]. Originally isolated from cultured porcine endothelial cells [1], there is also evidence that ET-1 is produced by smooth muscle cells in culture [16], indeed it has been subsequently shown that the proliferative responsiveness of human VSMCs correlates with ET receptor density [17]. ET-1-immunoreactivity is present in atherosclerotic plaque of human blood vessels [18,19] and there is abundant evidence that this peptide plays a promitogenic role in a variety of vascular diseases [2]. Apart from a potential role in the pathophysiology of coronary artery disease, ET-1 has been implicated in the pathogenesis of restenosis following PTCA. Raised circulating immunoreactive ET-1 has been demonstrated in patients following this procedure [3] and there is recent evidence of a time-dependent increase in ET-1, ET-3, ECE, ETA and ETB mRNA following balloon angioplasty in the rat [20]. This tissue-derived ET-1 may act on neighbouring receptors (both ETA and ETB), situated on adjacent VSMC cells, as well as those ‘downstream’ from atherosclerotic lesions or sites of vascular damage, for example distal segments of epicardial coronary arteries and myocardial microvessels [10].
There is experimental evidence that exogenous administration of ET-1 augments neointima formation after balloon catheter injury of the rat [4]; however, as these data are of necessity based on a very small number and have not yet been seen in any other species but rat the relevance for other animal models and especially so for man remains to be established. It has been reported that the nonpeptide ET receptor antagonist, SB 209670, reduces angioplasty-induced neointimal formation in the rat model of carotid artery injury [5]. Using this model, the ETA-selective antagonist, LU 135252, has recently been shown to reduce PTCA-induced neointima formation [7].
Whilst the rat carotid artery serves as a useful experimental model for studying the effects of vascular injury, a porcine coronary artery model has been described that mimics the pathology of human restenosis [21,22]. This model is closer to the human pathology then anything found in rodent models; however, it still has at least two methodological shortcomings. One of them may be the difference regarding arterial wound healing, scar formation, and thus remodelling and ‘recoil’ between pig and human coronary arteries [23]. The second difference of course relates to the difference in the health state: Whereas the pigs used in the present study and indeed in most other studies published with different drugs are relatively young and not suffering from atherosclerosis [24], the opposite holds true for patients. This may also account for at least some of the differences in arterial healing discussed above and certainly necessitates caution in any prognosis as to the relevance of the treatment for clinical use.
The main purpose of the present study was to determine the effect of the ETA antagonist, LU 135252, on PTCA-induced neointima formation in porcine coronary arteries. Apart from neointimal hyperplasia following balloon angioplasty there was a striking degree of adventitial remodelling one month post-PTCA, a phenomenon described following other forms of vascular injury [22]. Examination of the ‘neoadventitia’ of injured vessels revealed the presence of inflammatory cells (as described in porcine vein grafts, [25]), regions of neovascularization, similar to that observed following occlusion of the adventitial vasa vasorum [8] and a pronounced paravascular neoinnervation [26]. These regions exhibited ET-1-like immunoreactivity and/or ET ‘receptors’ and are the focus of ongoing studies.
The localization of immunoreactive ET-1 [14,18,19] and ET-1 receptors [8,10] on human epicardial coronary arteries has been reported. Here, using a combination of immunohistochemistry and in vitro autoradiography, we have identified ET-1 and its receptors within segments of injured and non-injured porcine coronary artery following balloon angioplasty.
Previous studies have shown that both mixed and ETA antagonists reduce angioplasty-induced neointima formation [4,5,27]. Here we present novel data indicating that there is a marked increase in ET receptor binding to medial vascular smooth cells following angioplasty and that this ‘upregulation’ of receptors is normalised by the ETA antagonist LU 135252. Both ETA and ETB binding sites (putative receptors) are present on the tunica media of porcine coronary arteries (ETA>ETB). In control animals the injured vessel segments exhibited a marked neointima and medial thickening 4 weeks post-PTCA. ETA/ETB binding to the tunica media of injured segments was significantly greater than binding to non-injured regions of the LAD. This increased binding is likely to reflect an increase in membrane ‘receptors’ since, although there was evidence of medial thickening, VSMC density (i.e. cells per mm2) of injured segments was unaltered. Interestingly, apart from reducing neointima formation following PTCA, the elevated level of medial ET binding to injured segments was normalized in animals receiving LU 135252. Since the mitogenic action of ET-1 on VSMCs has been shown to correlate with ET receptor density [17], ETA receptor blockade may also have an antiproliferative effect on those medial VSMCs involved in neointima formation.
ET binding, predominantly to ETA receptors, was also evident within the neointima of control animals and these regions of myointimal hyperplasia also demonstrated strong ET-1-like immunoreactivity (both associated with VSMCs). Restenosis, following balloon injury, involves the migration and proliferation of VSMCs from the media (contractile phenotype) to the intima (synthetic phenotype) with subsequent neointima formation [28]. Apart from ET-1 release, due to endothelial injury following PTCA, the rupture of the internal elastic lamina and mechanical stimulation of the media is also likely to lead to ET-1 release from VSMCs. Our results suggest that this peptide acts in an autocrine/paracrine fashion in this porcine model of coronary restenosis. Apart from any transient vasoconstrictor action, locally-generated ET-1 (from the endothelium and VSMCs) may also stimulate proliferation of VSMCs, leading to neointima formation and eventual vessel occlusion.
ET-1-stimulated VSMC proliferation and neointima formation has been shown to be blocked by ET-1 antagonists [29]. Early reports on the effect of ET antagonists on reducing experimental neointima formation utilised mixed ETA/ETB antagonists [5,30,31]and subsequent in vitro observations indicated that both receptor subtypes were associated with the ability of ET-1 to promote VSMC migration and mitogenesis [28,29]. Clearly, the potential of ET antagonist therapy in reducing neointima formation, following PTCA, has been suggested by various groups. However, it is not clear whether a single ET receptor subtype should be targeted, since experimental evidence to date indicates that the ‘vasculoprotective’ properties of ET antagonists may be species-dependent (ETA in the rat [6,30,31]; ETB in the rabbit [32,33]). Two recent reports on the effect of ETA receptor antagonists after neointima formation induced by stent implantation into pig coronary arteries support the finding of the present study, that in pigs ETA receptor blockade seems to be of primary importance. The compound ABT147627 was administered twice daily orally to pigs for 28 days in several doses and led to a decrease in neointima formation of about 35 percent independent of the dose tested [34]. In our laboratory a similar reduction was achieved in the same model, again applying 30 mg/kg/day LU 135252 for 4 weeks [24].
Our data presented here as well as the stent data discussed above may be more relevant to the human situation since they have been obtained using porcine epicardial coronary arteries, rather than rat/rabbit aorta or carotid artery. The histological changes induced by balloon angioplasty in the pig are similar to those seen in patients following PTCA [21,22]. However, as discussed above, the lack of atherosclerotic disease and possibly differences in arterial healing and remodelling needs to be taken into account. Nevertheless the predominance of ETA receptors in the media and neointima of injured segments of the coronary artery suggests that these represent sites where the ETA antagonist, LU 135252, acts to reduce neointima formation following PTCA and underscores the therapeutic potential of ETA antagonists in restenosis and other forms of vascular injury.
Time for primary review 21 days.
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Acknowledgements |
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MRD was supported by a project grant from the British Heart Foundation (PG 96035). We wish to thank Beate Degner, Elmar Forsch, Frank Hopfinger (animal studies), Peter Albert (morphometry) and Rosanna Gibbins (immunohistochemistry).
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