(Circulation. 1997;95:221-230.)
© 1997 American Heart Association, Inc.
Articles |
Endothelin-Converting Enzyme Expression in the Rat Vascular Injury Model and Human Coronary Atherosclerosis
the Third Department of Internal Medicine (T.M., H.K., K.M., Y.Y.), Department of Pathology (H.O., T.I.), Faculty of Medicine, University of Tokyo; The Biological Research Laboratories, Sankyo Co Ltd (M.T., K.S., K.T.), Tokyo; and Hachioji Medical Center of Tokyo Medical College (T.U.), Japan.
Correspondence to Hiroki Kurihara, MD, PhD, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail kuri-tky@umin.u-tokyo.ac.jp.
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Background Endothelin 1 has been implicated in various human diseases, including atherosclerosis. In this study, we examined the expression and localization of endothelin-converting enzyme-1 (ECE-1), the final key enzyme of endothelin 1 processing, in rat carotid arteries after balloon injury and in human coronary atherosclerotic lesions.
Methods and Results ECE-1 mRNA levels and ECE activity in rat balloon-injured arteries started to increase between 2 and 5 days after injury. The endothelin 1 content of tissue in injured arteries was concomitantly increased. Immunohistochemical staining located ECE-1 signals in endothelial cells in uninjured arteries, whereas ECE-1 immunoreactivity was detected in neointimal smooth muscle cells in injured arteries 5 to 14 days after balloon denudation. The size of the neointima was effectively reduced by phosphoramidon, an inhibitor of neutral metalloproteases, including ECE-1. In human coronary atherosclerotic lesions, intense ECE-1 immunoreactivity was detected in subsets of cells embedded in atheromatous plaque that correspond to smooth muscle cells and macrophages, as identified by staining for smooth muscle 
-actin and CD68 surface marker, respectively.
Conclusions The present study ascertained that ECE-1 is expressed in neointimal smooth muscle cells in rat balloon-injured arteries and in both smooth muscle cells and macrophages in human coronary atherosclerotic lesions. Blockade of ECE-1 was effective in reducing neointimal formation after balloon injury. Thus, ECE-1 may contribute to the process of injury-induced neointimal formation and atherosclerosis through the autocrine/paracrine effects of endothelin 1.
Key Words: atherosclerosis • endothelin • enzymes
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Endothelin 1 (ET-1) is a 21–amino acid peptide that is produced primarily by vascular endothelial cells.1 Three isopeptides (ET-1, ET-2, and ET-3) encoded by different gene loci2 share two distinct G protein–coupled receptors (ETA and ETB) with different affinities.3 4 In addition to exerting a vasoconstrictor effect, ET-1 also displays a diverse set of biological activities, such as stimulation of hormone release and modulation of central nervous activity,5 suggestive of it participating in the regulation of cardiovascular homeostasis and the pathogenesis of various diseases.6 7 8 Our recent studies9 10 on ET-1 knockout mice have revealed its novel role in embryogenesis, ie, ET-1 contributes to the development of specific neural crest–derived cell lineages giving rise to ectomesenchymal cells in the craniofacial and cardiovascular regions.
Recent findings have implicated ET-1 in the pathogenesis of atherosclerosis. ET-1 acts as a potent mitogen on many kinds of cells, including vascular smooth muscle cells.11 12 This mitogenic action is synergistic with the effect of other mitogens such as platelet-derived growth factor and epidermal growth factor.13 ET-1 also stimulates migration and matrix formation of vascular smooth muscle cells.13 14 The expression of ET-1 in cultured vascular endothelial cells is stimulated by several factors relating to the formation of atherosclerotic lesions, such as transforming growth factor-ß, interleukin-1, and oxidized LDL.15 16 17 In addition, ET-1 is extensively expressed in vascular smooth muscle cells and macrophages in human atherosclerotic plaques18 19 and rat atherosclerosis models of allografts.20 ET receptors are also detected in human atherosclerotic coronary arteries, especially in regions of neovascularization.21 22
The physiological role of ET-1 in experimental balloon injury models is also suggested by several previous studies. ET-1 and its receptors are highly upregulated in the rat and rabbit balloon models.23 24 25 Furthermore, neointimal hyperplasia after balloon injury is aggravated by ET-126 27 and is reduced by several ETA/ETB nonselective receptor antagonists,28 29 30 suggesting that ET-1 is involved in balloon-induced neointimal formation. However, controversy surrounds whether such an action is mediated by the ETA or ETB receptor. The ETA-selective antagonist BQ-123 is inactive in both the rat and rabbit balloon models.24 31 In contrast, neointimal formation can be attenuated by the ETA-selective antagonist BMS-182874.32 Thus, the exact pharmacological profile of the ET receptor antagonist, in terms of its receptor subtype selectivity, has not been clearly defined, and inhibition of ET-1 processing to reduce regional ET-1 levels may be another attractive therapeutic strategy for the disease processes underlying restenosis.
In ET-1 processing, conversion of the precursor polypeptide, big ET-1, to mature ET-1 is the final key step. This conversion has been postulated to be catalyzed by a putative ET-converting enzyme-1 (ECE-1).1 ECE-1 cleaves the nondibasic bond of Trp21-Val22. The activity of this enzyme is sensitive to phosphoramidon, a potent inhibitor of neutral metalloprotease.33 34 35 Because any alteration in ECE-1 activity has a potent and direct effect on ET-1 production, ECE-1 has been singled out as one of the major targets in therapeutic research on the ET system. Recently, ECE-1 was purified from rat lung36 and porcine aortic endothelial cells.37 Cloning and functional expression of ECE-1 cDNA revealed that ECE-1 is a membrane-bound metalloprotease homologous to neutral endopeptidase 24.11 and Kell blood group protein.38 39 ECE-1 mRNA has been shown to be expressed in various organs, including the lung, adrenal gland, heart, ovary, and testis, and most abundantly in vascular endothelial cells.38 39 Molecular characterization of ECE-1, therefore, would unveil much information on the pathophysiological role of ECE-1 and its substrate. In the present study, our goal was to further define the role of ECE-1 in the pathophysiology of vascular injury and atherosclerosis and to evaluate the value of ET-1 as a therapeutic target. For this purpose, we examined the expression and activity of ECE-1 both in rat models of neointimal formation after balloon denudation and in human directional coronary atherectomy samples. We also studied the effect of phosphoramidon, which can inhibit the activity of ECE-1, on neointimal formation after balloon injury.
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Rat Arterial Injury Model
The experimental protocol for the present study was designed in accordance with the "Guide for Animal Experimentation," Faculty of Medicine, University of Tokyo. Adult male Sprague-Dawley rats weighing 400 to 450 g were anesthetized with chloral hydrate (370 mg/kg IP). Balloon denudation of the left common carotid artery was performed as previously described.40 Briefly, a 2F arterial embolectomy balloon catheter (Baxter) was inserted into the left common carotid artery through the left external carotid artery. The balloon was inflated with saline at the proximal edge of the omohyoid muscle and withdrawn to the carotid bifurcation. This procedure was repeated a total of three times, and then the catheter was removed and the left external carotid artery was ligated. The right common carotid artery served as control. For RNA analysis and ECE assay, the left carotid arteries of sham-operated rats were also used for another control at day 14 to confirm the validity of the use of contralateral vessels.
For experiments on the effect of phosphoramidon, an osmotic minipump (Alzet model 2ML4; Alza Corp) containing phosphoramidon (10 mg·kg-1·d-1; Wako) dissolved in PBS was implanted subcutaneously into each rat 7 days before balloon denudation. This enabled administration of phosphoramidon for 28 days. Rats in whom osmotic minipumps containing PBS were implanted served as controls.
Rats were euthanatized with a lethal dose of anesthetic at various time points after balloon injury. The carotid arteries were perfused with PBS under 90 to 100 mm Hg, and the adventitia was removed for RNA analysis and ECE activity assay. Then, a 15-mm segment of the common carotid artery was excised from a point 10 mm proximal to the carotid bifurcation. In some experiments on ECE activity, the vessels were perfused with cold distilled water instead of PBS to remove endothelial cells.41 For RNA analysis and ECE activity assay, the isolated vessels were immediately frozen in liquid nitrogen. For immunohistochemical examination, the samples were perfused with 4% paraformaldehyde, fixed in 4% paraformaldehyde for 12 hours, and embedded in OCT compound (Miles Inc).
Human Specimens
Tissue specimens were retrieved from two patients (a 57-year-old male and a 64-year-old male) by directional coronary atherectomy. Both patients underwent atherectomy at the primary atherosclerotic lesion responsible for acute myocardial infarction that occurred 
2 weeks before the atherectomy. The specimens were fixed in 4% paraformaldehyde immediately after atherectomy and embedded in OCT compound (Miles Inc).
RNA Analysis
To obtain an RNA probe for rat ECE-1, a 732-bp fragment of ECE-1 cDNA was cloned from rat lung RNA by reverse-transcribed polymerase chain reaction. Sense (5'-GAGGATCTGGTGGACTCACTCTCC-3') and antisense (5'-CGTCTTGTTCAGGTAATAGTCTCT-3') primers were deduced from the literature sequence of rat ECE-1 cDNA.38 The amplified fragment lies within the sequence common to the two ECE-1 isoforms, ECE-1 (ECE-1b) and ECE-1ß (ECE-1a), which are produced through alternative splicing.42 43 This fragment was cloned into pCRII vector by use of a TA cloning kit (Invitrogen). A single-stranded, 32P-labeled antisense probe was synthesized with the use of linearized plasmid templates and SP6 polymerase. For the internal control probe, 32P-labeled antisense RNA was transcribed with the use of linearized pT7RNA18S plasmids containing an 80-bp sequence of 18S ribosomal RNA gene (Ambion). Total RNA samples were extracted from the common carotid arteries with the use of RNAzol (BIOTEX). The RNA samples were hybridized with ECE-1 RNA and internal control probes overnight at 45°C in 80% formamide, 100 mmol/L sodium citrate (pH 6.4), 300 mmol/L sodium acetate (pH 6.4), and 1 mmol/L EDTA. After treatment with RNase A plus RNase T1 (RPAII kit, Ambion), protected fragments (142 bp for ECE-1 and 80 bp for 18S ribosomal RNA) were separated by electrophoresis on 7% polyacrylamide/8 mol/L urea gels and visualized by autoradiography. The signal intensity was quantified with the use of an Autoimaging Analyzer BAS 2000 (FUJIX). To correct for differences in loading, the signal density of each RNA sample hybridized to the ECE-1 probe was divided by the density hybridized to the 18S ribosomal RNA probe.
ECE Assay
Preparation of rat arterial microsomes and measurement of ECE activity were performed as described previously.36 Excised samples were homogenized in a 10x volume of homogenization buffer (20 mmol/L Tris-HCl, pH 7.5; 5 mmol/L MgCl2; 0.1 mmol/L PMSF; 20 µmol/L pepstatin A; and 20 µmol/L leupeptin) by use of a Polytron homogenizer. The homogenates were centrifuged at 800g for 10 minutes, and the supernatant was further centrifuged at 100 000g for 45 minutes. The pellets were rinsed in homogenization buffer three times and recentrifuged. These pellets were then solubilized in homogenization buffer containing 0.5% (wt/vol) Triton X-100 and centrifuged at 100 000g for 60 minutes. The supernatant (6 µg protein) was then subjected to enzyme assay. The enzyme reaction was carried out at 37°C in 100 µL of assay buffer (20 mmol/L Tris-HCl, pH 7.0; 0.1% [wt/vol] bovine serum albumin; 20 µmol/L pepstatin A; 20 µmol/L leupeptin; and 20 µmol/L E-64) containing 0.1 µmol/L big ET-1 (rat, 1-39; Peptide Institute). After 1 hour of incubation, the reaction was stopped by the addition of an equal volume of 5 mmol/L EDTA. The concentration of mature ET-1 was determined by use of an ELISA. Tissue ECE activity was expressed as the amount of ET-1 generated per 6 µg protein of tissue extract in this assay.
ELISA
For measurement of the ET-1 levels, we used a sandwich ELISA as previously described.44 This assay system also detects ET-2, and the cross-reactivity is 160%. The cross-reactivity of big ET-1 is 0.15%.
Immunohistochemistry
The samples embedded in OCT compound were serially cut into 6-µm frozen sections by use of a cryostat. Slide-mounted tissue sections were washed with PBS, treated with 0.3% H2O2 in methanol, preincubated with goat nonimmune serum, and incubated with mouse anti-rat ECE-1 monoclonal antibody AEC27-121 (IgG1)38 or anti-rat/human ECE-1 monoclonal antibody AEC32-236 (IgG1)45 for 16 hours at 4°C. These antibodies recognize both ECE-1/b and ECE-1ß/a.42 The sections were then incubated with biotinylated goat anti-mouse IgG (IBL, Inc) for 60 minutes at 37°C. After washing, the sections were treated with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC kit, Vector Labs) and developed with 0.004% H2O2 and 0.02% diaminobenzidine tetrahydrochloride. Next, the slides were counterstained with hematoxylin. Sequential sections were stained with anti-smooth muscle -actin monoclonal antibody (Boehringer Mannheim), anti–von Willebrand factor polyclonal antibody (Dako), or anti-CD68 monoclonal antibody (Zymed) to identify smooth muscle cells, endothelial cells, and macrophages, respectively. For the negative control, samples were stained with preimmune serum instead of the primary anti-rat ECE-1 antibody.
For double-label immunostaining, human tissue sections were first treated with AEC32-236 (IgG1) followed by incubation with Texas red–labeled horse polyclonal anti-mouse IgG (Vector Labs) for 1 hour at 37°C. Then, the sections were treated with anti–smooth muscle -actin monoclonal antibody or anti-CD68 monoclonal antibody for 1 hour at 37°C and stained with fluorescein-labeled horse anti-mouse IgG (Vector Labs) for 1 hour at 37°C. The samples were examined under an MRC1024 confocal laser microscope (BioRad).
Morphometric Analysis
We evaluated the effects of phosphoramidon on neointimal formation by measuring the size of the intimal lesion. The middle third of the left carotid artery was embedded in paraffin, and multiple 6-µm cross sections were stained with hematoxylin and eosin. After the sections were photographed, images were scanned and analyzed with the use of NIH Image 1.58 software (National Institutes of Health). Then, the areas of the neointima and the media were calculated. Three or four portions of each sample were analyzed, and the sections with the largest neointima-to-media ratios were subjected to statistical analysis.
Materials
Chemicals were purchased from Sigma Chemical Co unless otherwise specified.
Statistical Analysis
All quantitative values were expressed as mean±SEM. The intergroup difference in ECE activity was evaluated with the use of one-way ANOVA. For other comparisons, the Student's t test was used to determine significant differences. A value of P<.05 was considered significant.
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Expression of ECE-1 in Rat Normal and Balloon-Injured Arteries
An important starting point in the present study was an examination of ECE-1 mRNA levels in the balloon-injured arteries. As shown in Fig 1
, ECE-1 mRNA levels appeared to decrease transiently 6 hours and 2 days after injury. Thereafter, ECE-1 mRNA levels increased and reached 5-fold the levels in the contralateral vessels or sham-operated left carotid arteries at day 14. Three independent experiments yielded similar results.
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To examine whether the changes in ECE-1 mRNA levels were correlated to increases in enzyme activity, we measured ECE activity in microsomes prepared from the balloon-injured and control arteries. The ECE activity was equated as being the level of ET-1 converted from the polypeptide substrate precursor, big ET-1. Because ECE-1 is the major isoenzyme responsible for the cleavage of big ET-1 at the neutral pH (discussed later), the ECE activity is thought to reflect the activity of ECE-1. As shown in Fig 2, ECE activity increased up to 1.8-fold at day 14. ECE-1 activity seemed to decrease transiently at day 2, although this was not statistically significant. Phosphoramidon was found to completely inhibit this enzyme activity (data not shown). Because the increase in protein content in the injured arterial segment at day 14 was 2-fold that of the uninjured arterial segment or sham-operated left carotid artery, the ECE activity per segment represents a >3-fold increase in the injured artery at day 14. This time course of the ECE activity was quite similar to that of ECE-1 mRNA levels, suggesting that changes in ECE activity reflect ECE-1 mRNA levels.
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P<.005 vs sham-operated rats.
Next, it was examined whether arterial injury was a cause of increases in the tissue levels of ET-1. At day 14, ET-1 levels in the injured artery were 2-fold those in the uninjured artery (Fig 3). Although this increase may be due to a rise in ET-1 gene expression, this result is consistent with the increase in ECE-1 expression and ECE activity in the injured artery.
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To investigate the source of ECE-1 expression in the uninjured and injured arteries, we examined the effect of cold distilled water perfusion, which can remove endothelial cells, on ECE activity. In the uninjured artery, water perfusion significantly reduced ECE activity by 55% (Fig 4), suggesting that endothelial cells are the main eliciting cells of ECE-1 in the uninjured artery. In contrast, water perfusion had no significant effect on ECE activity in the injured artery (Fig 4). Clearly, this result indicates that the increased ECE-1 expression is attributable to non–endothelial cell components in the injured artery.
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Localization of ECE-1 Immunoreactivity in Rat Normal and Balloon-Injured Arteries
To locate ECE-1 signals in normal and balloon-injured arteries, we performed immunohistochemical staining using an anti–ECE-1 monoclonal antibody. Two antibodies, AEC27-121 and AEC32-236, with different epitope specificities gave the same results. In the uninjured artery, staining for ECE-1 was restricted to the endothelial cell monolayer, which was identified by staining for von Willebrand factor (Fig 5A, 5B, and 5C). No specific staining for ECE-1 was seen in the media or adventitia. At day 2 after balloon injury, no staining for ECE-1 was detected in the denuded artery (Fig 5D). ECE-1 staining was then observed in the neointima at day 5 (Fig 5E and 5F). At day 14, the neointima became markedly thick and intense, with ECE-1 staining being detected in the entire neointima but not in the media (Fig 6A, 6C, 6D, and 6F). Staining for smooth muscle -actin enabled identification of smooth muscle cells as the major cell component of the neointima and media (Fig 6B and 6E). It is inferred from these results that the increase in ECE-1 expression in the injured artery is attributable to the neointimal smooth muscle cells, and the expression of ECE-1 may depend on the phenotype in smooth muscle cells.
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Effect of Phosphoramidon on Balloon-Induced Neointimal Formation
To further investigate the pathophysiological significance of ECE-1 in the balloon model, we examined the effect of phosphoramidon, a metalloprotease inhibitor that can block the activity of ECE-1, on neointimal formation after balloon injury. After administration of phosphoramidon 10 mg·kg-1·d-1 for 21 days, production of ET-1 was greatly suppressed in uninjured and injured arteries (Fig 3
), indicating that this dose of phosphoramidon is sufficient to inhibit ECE-1 in the arterial wall.
The sizes of the neointima and media of the balloon-injured artery were compared in phosphoramidon-treated rats and PBS-treated control rats. In the present study, most of the control rats exhibited severe responses to balloon injury, with an average neointima-to-media ratio of 1.77 (Fig 7C). Treatment with phosphoramidon did not change the size of the media (control rats [n=8], 0.213±0.008 mm2; phosphoramidon-treated rats [n=10], 0.235±0.010 mm2) (Fig 7A) but significantly decreased the size of the neointima (control rats, 0.377±0.025 mm2; phosphoramidon-treated rats, 0.193±0.017 mm2) (Fig 7B) and consequently the neointima-to-media ratio by 50% (control rats, 1.77±0.11; phosphoramidon-treated rats, 0.91±0.13) (Fig 7C). Typical examples of the response to balloon injury in control and phosphoramidon-treated rats are shown in Fig 8A and 8B, respectively. No abnormal findings were observed in contralateral right carotid arteries of either group of rats (data not shown).
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Expression of ECE-1 in Human Coronary Atherosclerotic Lesions
To investigate ECE-1 expression in human atherosclerosis, we stained directional coronary atherectomy samples for ECE-1. ECE-1 staining was detected in both samples from the two patients. ECE-1 immunoreactivity was predominantly localized to hypercellular areas (Fig 9A and 9C). Most of the cells in these areas were positive for smooth muscle -actin (Fig 9B), suggesting that ECE-1 was expressed mainly by smooth muscle cells. Cells positive for ECE-1 staining seemed scattered in comparison to smooth muscle -actin–positive cells. In addition, ECE-1 was expressed in the macrophage-rich region identified by anti-CD68 monoclonal antibodies (Fig 10).
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Cells expressing ECE-1 were further characterized by double immunostaining. Double staining for ECE-1 and smooth muscle -actin demonstrated the expression of ECE-1 in smooth muscle (Fig 11A, 11B, and 11C). ECE-1 immunoreactivity was also colocalized with staining for CD68 surface antigen, indicating that ECE-1 is also expressed in macrophages (Fig 11D, 11E, and 11F).
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Discussion |
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The present study was designed to assess the expression of ECE-1 in the rat balloon injury model and human coronary atherosclerosis. Although the pathophysiological characteristics of the rat balloon injury model differ in many respects from those of human atherosclerosis, the animal model reflects some aspects of atherosclerosis, especially in terms of the way smooth muscle cells contribute to neointimal formation.46 Thus, an examination of ECE-1 expression in the rat model in concert with human atherosclerosis is expected to substantiate and build on existing knowledge about the pathophysiological role of the ET system.
Two types of ECE isoenzymes, ECE-1 and ECE-2, have been identified.47 ECE-1 is associated with plasma membranes and has a neutral pH optimum, whereas ECE-2 is an intracellular enzyme with an acidic pH optimum.47 In addition, ECE-1 is abundantly expressed in endothelial cells and other ET-1–producing cells compared with ECE-2.47 Thus, ECE-1 is thought to be mainly responsible for the cleavage of big ET-1. ECE-1 has two subtypes, ECE-1 (ECE-1b) and ECE-1ß (ECE-1a), which are encoded by the same gene and are produced through alternative splicing.42 43 These two ECE-1 subtypes differ only by their N-termini and share the same 726 C-terminal residues containing the catalytic domain.42 43 Although these two ECE-1 isoenzymes exhibit distinct expression patterns in various tissues, no functional difference between them has been proved. In the present study, RNase protection assay and immunohistochemistry did not distinguish these isoenzymes. Thus, the ECE-1 activity in vascular wall was grossly evaluated as a combination of the two subtypes, ECE-1/b and ECE-1ß/a, in the present study.
ECE-1 in Rat Balloon Injury Model
In uninjured rat common carotid arteries, ECE-1 was detected only in endothelial cells. After endothelial denudation by balloon injury, ECE-1 was extensively detected in neointimal smooth muscle cells. The appearance of ECE-1 immunoreactivity started between days 2 and 5, when smooth muscle cell migration to the neointima is initiated.26 The levels of ECE-1 mRNA per equal RNA contents and ECE activity per equal protein contents was significantly increased compared with those of control vessels 14 days after injury. Because little endothelial regeneration was detected at this time and ECE activity of the injured arteries was not affected by cold water perfusion, the major source of ECE-1 in the injured arteries appears to be neointimal smooth muscle cells. Taking into account the increased mass of neointimal cells and extracellular matrices, a >2-fold increase in ECE-1 expression and enzyme activity per arterial segment is likely to be attained. Correspondingly, the ET-1 content of tissue in the injured arteries is increased compared with that of the uninjured arteries. The pattern of ECE-1 induction in the injured vessels is reminiscent of that of ACE induction reported by Rakugi et al.41 They demonstrated that ACE expression and enzyme activity are increased by 2-fold in smooth muscle cells in the neointima induced by balloon injury. Although the molecular characteristics are distinct between the two converting enzymes, similar mechanisms may act in inducing these enzymes in neointimal smooth muscle cells.
In contrast to the extensive ECE-1 expression in neointimal smooth muscle cells, no detectable staining for ECE-1 was observed in medial smooth muscle cells. However, residual ECE activity after cold water perfusion and ECE-1 gene expression in endothelium-denuded arteries at day 2 after balloon injury suggest the possible low expression of ECE-1 in medial smooth muscle cells. This speculation is consistent with previous reports48 49 that phosphoramidon-sensitive ECE activity is also present in noncultured vascular smooth muscle. An interesting result revealed in the present study, however, was that ECE-1 expression is much higher in intimal smooth muscle cells than in medial smooth muscle cells and may be related to phenotypic changes of smooth muscle cells from the contractile type to the synthetic type.
Recently, Wang et al50 reported that rat balloon injury results in the induction of ECE-1 mRNA by semiquantitative polymerase chain reaction. In contrast to our results, they demonstrated only a transient increase in ECE-1 mRNA levels within 24 hours after balloon injury. In the present study, histological examination showed extensive deendothelialization after balloon injury, and ECE-1 mRNA seemed rather decreased. Furthermore, ECE-1 was not detected in smooth muscle cells within 2 days in our experiments using immunohistochemistry. Although Wang et al50 did not show histological and immunohistochemical data, milder balloon denudation may cause the increase in ECE-1 mRNA in residual endothelial cells 6 to 24 hours after injury, and extensive balloon injury may be required for ECE-1 gene induction in smooth muscle cells.
The role of ECE-1 in neointimal formation was further confirmed by the beneficial effect of phosphoramidon. Phosphoramidon is an inhibitor of neutral metalloprotease, and ECE-1 is one of its major targets.33 34 35 In the present study, the in vivo administration of phosphoramidon could effectively suppress the regional production of ET-1 in the arterial wall. Significant reduction of neointimal size after balloon injury by phosphoramidon strongly suggests that ECE-1 may contribute to neointimal formation through an increase in ET-1 production.
ECE-1 in Human Atherosclerosis
Although human atherosclerosis is distinct from the rat balloon injury model, they share a common feature of neointimal smooth muscle proliferation. In human coronary atherosclerotic lesions, an intense ECE-1 immunoreactivity was detected in smooth muscle cells identified by anti–smooth muscle -actin. This result apparently corresponds to ECE-1 expression in neointimal smooth muscle cells of the rat injury model. In addition, ECE-1 is also colocalized to CD68 immunoreactivity, indicating that ECE-1 is produced by macrophages as well as smooth muscle cells in atherosclerotic lesions. These patterns of ECE-1 localization in human coronary atherosclerotic lesions resemble those of ET-1 localization described in previous studies. Lerman et al18 demonstrated that plasma ET-1 levels are correlated with the severity of human atherosclerosis and that ET-1 expression is detected in vascular smooth muscle cells in atherosclerotic lesions. More recently, Zeiher et al19 reported that ET-1 immunoreactivity is localized in macrophages and smooth muscle cells of active coronary atherosclerotic plaque. These findings suggest that components of the ET system are coexpressed in the same cell populations present in atherosclerotic plaque. In the process of ET-1 production, ECE-1 is an essential enzyme catalyzing the final conversion of the precursor to mature ET-1. A rational conjecture would be that ECE-1 is coexpressed with the ET-1 gene to produce mature ET-1 peptide.
The mechanisms of ECE-1 gene expression coupled with ET-1 expression remain unknown. Previous in vitro experiments have shown that ET-1 production is induced by several factors related to vascular injury and atherosclerosis in smooth muscle cells and macrophages. Hahn et al51 demonstrated that ET-1 expression in cultured vascular smooth muscle cells is induced by transforming growth factor-ß, platelet-derived growth factor, and angiotensin II, which are regarded as likely mediators of neointimal formation.46 It was reported that lipopolysaccharides can induce ET-1 production in macrophages.52 Although these mechanisms of ET-1 induction are active in the in vivo atherosclerotic process, it is of interest to determine whether these stimulators can also induce ECE-1 expression directly or whether induced ET-1 production can upregulate the expression of its processing enzyme, ECE-1.
Clinical Implications
Several studies support the idea that ECE-1 activity may regulate the tissue and plasma levels of ET-1. Plasma molar levels of big ET-1 are much higher than those of ET-1 in humans and animals.53 54 In rats with congestive heart failure, plasma ET-1 levels are significantly increased, whereas plasma big ET-1 levels remain unaffected.55 These findings are indicative of the majority of big ET-1 being released from vascular endothelial cells (and other possible ET-secreting cells) without being converted to ET-1, and thus ECE-1 activity may be correlated with pathological states. When big ET-1 is administered intravenously, it is converted to ET-1 and produces a pressor effect that can be blocked by phosphoramidon.33 Thus, levels of ECE-1 expression and activity may contribute to the regulation of regional ET-1 production and its effects on target cells.
Several things implicate ET-1 in the pathogenesis of human atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Although ET-1 is detected only in endothelial cells in the nonatherosclerotic arterial wall, it is extensively expressed in vascular smooth muscle cells and macrophages in human atherosclerotic plaques.18 19 ET receptors are also detected in human atherosclerotic coronary arteries, especially in regions of neovascularization.21 22 Although no definitive data have been provided on the involvement of ET-1 in restenosis after PTCA in human study, several experimental findings have suggested that ET-1 plays a potential role in neointimal formation after balloon injury. Several ETA/ETB nonselective receptor antagonists have been reported to reduce balloon-induced neointimal formation,28 29 30 but the exact pharmacological profile of the ET receptor antagonist, in terms of its receptor subtype selectivity, has not been clearly defined.24 31 32 Such a controversy would be circumvented by suppressing the production of ET-1 with an ECE-1 inhibitor. In fact, the present study demonstrates that blockade of ECE-1 results in a significant reduction in balloon-induced neointimal formation. Taken together with the increased ECE-1 expression in the rat balloon injury model and human coronary atherosclerotic lesions, the present study would purport the idea that ECE-1 can be a potential target of therapy for atherosclerosis and angioplasty restenosis.
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Acknowledgments |
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This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan; the Sankyo Foundation; Kowa Life Science Foundation; Japan Cardiovascular Research Foundation; Kanae Foundation of Research for New Medicine; TMFC; and the Ryoichi Naito Foundation for Medical Research. We thank Chie Fujinami and Shouichi Moro for their technical assistance.
Received May 9, 1996; revision received August 19, 1996; accepted August 24, 1996.
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