(Hypertension. 1997;30:272-277.)
© 1997 American Heart Association, Inc.
Articles |
Early Induction of Angiotensin I–Converting Enzyme in Rat Carotid Artery After Balloon Injury
From the Departamento de Farmacologia, Facultad de Farmacia, Universidad Complutense, Madrid, Spain (M.S.F.-A.); Hoechst Marion Roussel, Pharma Forschung HMR TD, Cardiovascular Agents, Frankfurt am Main, Germany (P.A.M., P. van E., D.T., B.A.S.); and the Department of Clinical Pharmacology and Toxicology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Germany (I.L., M.P.).
Correspondence to Prof Martin Paul, Department of Clinical Pharmacology and Toxicology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail paul{at}ukbf.fu-berlin.de
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Abstract |
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Abstract Several studies have demonstrated the effectiveness of angiotensin I–converting enzyme (ACE) inhibitors in preventing the neointima formation found after denudation of the rat carotid artery by balloon injury. The aim of the present study was to determine the role of ACE in this model and to compare the treatment with the ACE inhibitor ramiprilat with that with the angiotensin II antagonist HR 720. The endothelial layer of the left carotid artery was removed using an inflated balloon catheter. Injured and control vessels were both submitted to histomorphological analysis and DNA content quantification at 2, 4, 6, 8, 12, and 14 days after injury. Evaluation of neointima thickening demonstrated a slow but steady increase of neointima that was significant after day 6 and reached 30% of the lumen in 2 weeks. This was paralleled by an increase in DNA content, which was significant 4 days after injury. ACE mRNA levels were quantified by polymerase chain reaction after reverse transcription. Measurement of ACE mRNA levels revealed a significant upregulation 2 and 8 days after injury, with no significant difference when compared with control tissue at later time points. ACE activity was also significantly enhanced at 2 and 8 days after injury, with no significant difference when compared with control tissue at later time points. In addition, the treatment with ramiprilat was more efficient in reducing neointima formation than that with HR 720. These data underlie the role of ACE in this model of restenosis. The early induction of ACE expression after endothelial injury but before significant changes in the vessel structure suggests that ACE activity might be one of the mechanisms that trigger neointima formation in the rat.
Key Words: angiotensin-converting enzyme inhibitors • angiotensin-converting enzyme • balloon injury • polymerase chain reaction
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Introduction |
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Endothelial injury induced by a balloon catheter results in a number of trophic changes in the vessel wall, such as myointimal hyperplasia. After removal of the endothelial cell layer, quiescent medial smooth muscle cells start to proliferate and then migrate into the lumen to form a neointima, where they further proliferate and release extracellular matrix components.1 This process is thought to be one of the underlying mechanisms of restenosis in patients that occurs in 25% to 35% of dilated vessels 6 months after angioplasty.2
Several data strongly suggest the contribution of Ang II to myointimal hyperplasia. ACE inhibitors1 3 4 5 6 and AT1-receptor antagonists7 8 9 10 have been shown to decrease the occlusive lesion in a variety of species. The beneficial effects of these drugs in this experimental model have been related primarily to the trophic effects of Ang II.11 In fact, Ang II can stimulate extracellular matrix production,12 smooth muscle cell proliferation,13 and migration.14 When administered in vivo, Ang II stimulates smooth muscle cell proliferation in the media of rat aorta and carotid artery.6 15 Also, an activation of the renin-angiotensin system in this model has been previously suggested, based on the enhancement of angiotensinogen mRNA expression16 and upregulation of AT117 and AT218 receptor expression in balloon-injured rat aorta.
However, the origin of Ang II that may participate in the myointimal proliferative response to injury remains an open question. Contradictory results showed equal19 or higher20 ACE activity in the neointima 14 days after injury. At this time point neointima formation is already completed, and it is not possible to conclude whether ACE present at the layer is the cause or the consequence of neointima formation.
The aims of the present study therefore have been (1) to characterize the time course of ACE expression and activity during lesion initiation and development; (2) to determine whether ACE is formed locally at the lesion site, measuring ACE mRNA expression by a quantitative PCR assay as well as ACE activity; and (3) to determine whether a treatment with the ACE inhibitor ramiprilat or with a novel AT1 antagonist, HR 720, is efficient in reducing the occlusive process.
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Methods |
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Arterial Injury Model
Endothelial denudation was performed in the left common carotid artery of 3-month-old male Sprague-Dawley rats (300 to 400 g). Under halothane anesthesia, the distal left common carotid artery and the region of the bifurcation were exposed and a Fogarty 2F balloon catheter was introduced through the external carotid artery and advanced into the aorta. Then the balloon was inflated until it reached a diameter of 2 mm and was then moved back to the carotid bifurcation three times. We documented histologically that this procedure effectively denuded the endothelium without damaging the lamina elastica interna. Then the balloon catheter was removed, the artery was ligated, and the wound was closed. At the different time points after deendothelialization, rats were anesthetized (urethane 1.3 g/kg IP), treated with papaverin (6 to 30 mg · kg-1 · min-1 as an infusion in the jugular vein for 5 minutes) and heparin (1000 U/kg as an intravenous bolus), and thoracotomized. All animals were perfused via the left ventricle with phosphate buffer for 5 minutes at 80 mm Hg. Both the right (not deendothelialized=control) and the left (deendothelialized=injured) carotid arteries were dissected and the adventitia grossly peeled off and divided into 5-mm-long pieces. The middle piece was immersed in 2.5% glutaraldehyde and processed for histological examination and morphometric assessment. The other pieces were stored at -80°C and used later for determination of DNA content, RNA isolation, and ACE activity. After they were embedded in Epon 812, semi-thin sections of 2 µm were stained with toluidine blue and basic fuchsin and evaluated morphometrically. Cross-sectional areas of neointima, media, and lumen (µm2) were determined with a Bioquant morphometry system.
DNA Quantification
DNA quantitation was performed fluorometrically in crude
homogenates of the carotid arteries according to the method
of Labarca and Paigen.21 Briefly, arteries were frozen in
liquid nitrogen, powdered, and resuspended in phosphate buffer. After
addition of the fluorochrome Hoechst 33258 (2 µg/mL), samples
were measured at a wavelength of 450 nm.
RNA Extraction and RT-PCR Analysis
Total RNA was isolated with the guanidinium isothiocyanate
method,22 precipitated, dissolved in water, and quantified
twice by absorbance at 260 nm. The intactness of the RNA was checked on
ethidium bromide–stained agarose minigel. ACE expression was assessed
by RT-PCR, as previously described.23 Twenty-five PCR
amplification cycles were run in the following order: 30 seconds at
94°C for denaturation, 1 minute at 55°C for annealing, and 1 minute
at 72°C for primer extension, with an additional 7 minutes at 72°C
at the end for final extension. After hybridization with a
32P-labeled specific primer, blots were exposed for 2 hours
to an imaging plate (Fuji Photo Film Co), and
autoradiograms were scanned with a computer-based
imaging system (Fujix Bas 2000, Fuji Photo Film Co). Quantification of
ACE mRNA was performed in the presence of a defined concentration of
ACE-cDNA mutant as internal standard, as previously
described.23 One microgram of reverse-transcribed RNA was
mixed with the appropriate amount of mutant ACE cDNA, ranging from 20
to 1 pg, in which neither the endogenous nor the mutant ACE
would completely suppress their counterparts. To create a standard
curve for the estimation of endogenous ACE, this mixture
was then serially diluted 1:2 for five times. The standard curve was
then used for quantification, minimizing in this way sample-to-sample
variations. Results are expressed as picograms of ACE mRNA per
microgram of total RNA.
ACE Activity Determination
For ACE activity determination the carotids were
homogenized in 0.3% Triton solution, sonicated, and
centrifuged at 20 000g for 20 minutes at 4°C.
Tissue ACE activities were determined using a modification of the
fluorometric method according to Depierre and Roth,24 with
carbobenzoxyphenylalanyl-histidyl-leucine (Z-Phe-His-Leu) used as
substrate. Protein content of the tissue homogenates was
analyzed according to the method of Lowry et al.25
The data are expressed as nanomoles of histidine-leucine (His-Leu)
per milligram of protein.
Drugs
Ramiprilat,
2-N-((S)-1-carboxy-3-phenylpropyl)-L-alanyl-(1S,3S,5S)-2-azabicyclo(3.3.0)octa-ne-3-carboxylic
acid, is the active metabolite of ramipril due to deesterification.
Ramiprilat inhibits ACE with a Ki
value of 7 pmol/L. It is both a slow- and a tight-binding
inhibitor, and the mode of action of inhibition is fully
competitive. Ramipril lowers blood pressure in various models of
hypertension and improves states of acute cardiac failure mainly by
suppression of Ang II formation.
HR 720, dipotassium 2-butyl-4-(methylthio)-1-((2'-((((propylamino)carbonyl)amino)sulfonyl)(1,1'-biphenyl)-4-yl)-1H-imidazole-5-carboxylate, is a new, nontetrazolic AT1 selective and orally active Ang II receptor antagonist. HR 720 induces a displacement of 125I–Ang II binding to AT1 and AT2 receptors from membrane preparations with an IC50 of 0.48±0.01 and 920±293 nmol/L, respectively. In isolated guinea pig ileum, HR 720 induces a concentration-dependent inhibition of Ang II response with an IC50 of 5 nmol/L. Similarly, HR 720 (0.1 to 1 nmol/L) produces a concentration-related displacement to the right of the Ang II concentration-response curve on isolated rings of rat portal vein. This effect is insurmountable in nature.
Pharmacological Studies
Ramiprilat and HR 720 (Hoechst) were dissolved in a
solution of 50% dimethyl sulfoxide and 50% water and continuously
administered via an Alzet pump (2ML4) implanted subcutaneously in the
back region of the neck immediately after
deendothelialization. Three doses of
ramiprilat were administered: 0.1 (n=10), 0.31 (n=12), and
1. 0 (n=12) mg · kg-1 ·
d-1. Since the pump starts delivering its
content at a constant rate 4 hours after implantation, a single dose
was given intraperitoneally as a bolus 30 minutes
before the lesion was made to cover the therapeutic gap. Control groups
received just the solvent in the same way. Because of the poor
solubility of ramiprilat, a higher dose of this compound
(3.1 mg · kg-1 ·
d-1) could not be administered with one Alzet
pump. Consequently, two Alzet pumps (2ML2) were implanted
subcutaneously in the back region of the rats (n=14). A new group of
control animals (n=14) receiving solvent via two Alzet pumps was also
investigated. In all animals, the two pumps were replaced by new ones
after 14 days. Weight gain of the animals was measured during the
treatment. Twenty-one days after induction of the lesion the animals
were killed by a blow on the head. Two doses of HR 720 were
administered: 3 mg · kg-1 ·
d-1 (n=12) and 10 mg ·
kg-1 · d-1
(n=12). Results are expressed as mean±SEM. Differences between groups
were compared using one-factor ANOVA or ANOVA for repeated measures as
appropriate. A value of P<.05 was considered statistically
significant.
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Results |
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Injured and control vessels were subjected to histomorphological analysis at 0, 2, 4, 6, 8, 10, 12, and 14 days and at 3 months after injury. The lesion was characterized by moderate platelet adhesion on the denuded intima without thrombus formation and vascular smooth muscle cell migration and proliferation with extracellular matrix deposition (Fig 1
). Morphometric evaluation of
neointima thickening demonstrated that there was a slow and
steady increase of the area of the new layer, which was significant
from day 6 (Fig 2). Two weeks after
injury, neointima reached 30% of the lumen and remained
stable for up to 3 months. No variations of the cross-sectional area of
the media could be observed for up to 3 months after denudation. In
addition to morphometry, cell proliferation was quantified by
fluorometric evaluation of DNA content at 0, 2, 4, 6, 8, 10, 12, and 14
days and at 3 months after injury. DNA showed a slow and steady
increase that was significant from day 4 after injury, reaching a
maximum at day 21, after which it remained stable (Fig 3).
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Because of the low amount of RNA that is extracted from blood vessels,
ACE mRNA was measured by quantitative RT-PCR by amplification of 1 µg
total RNA from carotid arteries. A linear increase in the amount of
input RNA showed a linear increase in amplification (results not
shown). To ensure that the sample was not contaminated with DNA, PCR
amplification was performed avoiding the RT reaction (results not
shown). The yield of RNA per gram of tissue was approximately three
times higher (P<.05) in injured vessels
(Table).
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ACE expression showed a significant upregulation at early time points
after denudation. ACE mRNA levels were significantly higher
(P<.05) in the injured carotid artery at 2 and 8 days after
balloon catheterization compared with the contralateral
vessel. In contrast, at 14 and 21 days after injury no differences
between both vessel groups could be observed (Fig 4).
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ACE activity was measured at 2 and 8 days after injury, which
were the time points of the highest ACE mRNA concentrations, and at day
14. Injured vessels were compared with their contralateral controls
(without endothelium) at day 0, showing a significant
upregulation of ACE activity at 2 and 8 days after the lesion
(P<.001) (Fig 5). Delivery of
ramiprilat elicited a dose-related reduction of
neointima area (Fig 6A) and
DNA content in the injured arteries (Fig 6B). The threshold dose in
both cases was 0.31 mg · kg-1 ·
d-1, and maximal effects were obtained with
the dose of 3.1 mg · kg-1 ·
d-1. Only the highest dose led to a
significant reduction of weight gain in the animals and the area of the
media (results not shown). Delivery of HR 720 elicited a dose-related
reduction of neointima area (Fig 7) without affecting the cross-sectional
area of the media. However, when comparing the effects of both
treatments, ramipril was more efficient in reducing
neointima area than HR 720.
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Discussion |
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In this study, we demonstrate for the first time an induction of ACE at early time points after balloon catheter denudation of rat carotid arteries but before significant changes in the vessel wall structure. In addition, delivery of the ACE inhibitor ramiprilat or the AT1 antagonist HR 720 caused a dose-dependent inhibition of neointima formation without affecting other vascular layers.
Most studies concerning the model of myointimal proliferation after balloon catheter deendothelialization focus on the events that happen 2 weeks after injury.3 4 5 6 7 8 9 10 At this time point, however, neointima is already formed and it is not possible to conclude whether growth factors present at the injury are responsible for its formation or whether they are produced by the new layer itself. In our opinion, the early periods after deendothelialization are crucial for the understanding of the mechanisms that trigger neointima proliferation. In this study, we show important time course differences in ACE mRNA levels and activity during the development of the occlusive process. ACE expression and activity determinations both show that ACE is already upregulated 2 days after injury and that it remains induced until 8 days after endothelial damage, suggesting that Ang II may be produced locally at the lesion. The discrepancies in the degree of upregulation at the mRNA and enzyme level could reflect additional regulatory mechanisms such as pre- or posttranslational modification. No molecular or biochemical differences could be detected 14 days after denudation. Similar results have been described by Viswanathan et al,17 who showed that ACE activity was not different in the aortas of injured and control animals 15 days after injury. To the contrary, Rakugi et al20 have demonstrated an increased ACE activity at the same time point, specifically restricted to the neointima. It is interesting to note that ACE is already upregulated at the injury before the beginning of morphometric changes in the vessel wall structure. Significant increases in DNA content cannot be detected until day 4 after injury, whereas the increase in neointima area is significant only after day 6. These results are consistent with the description by Clowes et al1 of the cellular events that take place during neointima formation. After endothelial removal, there is proliferation of quiescent smooth muscle cells into the media (day 4) and then a subsequent migration of these cells into the lumen to form the neointima (day 6), where they further proliferate and release extracellular matrix until the whole process is completed (day 14). The early upregulation of ACE and its subsequent downregulation after completion of this process suggest that Ang II formation in the media in the first phase may be one of the mechanisms that trigger the initiation and early development of neointima formation, in addition to other growth factors or modulators that have been proposed to participate in the complex processes involved in neointima formation. ACE is well known to be present in the endothelial layer and adventitia,26 and some authors have also suggested the existence of ACE in the media.23 27 28 In our opinion, the role of a medial ACE is not relevant when the endothelium is present. However, the removal of this layer results in a quick upregulation of ACE in the media before changes in the vessel structure can be detected. Consequently, because of an enhanced ACE activity at the site of the lesion, there might be an imbalance between an increased Ang II formation and a lack of endothelium-derived growth inhibitor production that could contribute to the proliferation of smooth muscle cells and neointima formation. The importance of Ang II as a growth factor or modulator in this model has also been demonstrated by the fact that neointima formation is inhibited by blocking AT1 receptors with losartan.7 8 9 Similarly, in this study we demonstrate that the novel AT1-receptor antagonist HR 720 induces a dose-dependent inhibition of neointima formation without affecting other vascular layers to a degree similar to that of treatment with ramiprilat, which confirms that Ang II production is a crucial event for neointima formation. Contradictory results with ACE inhibitors have been obtained by other investigators. Positive results in reducing neointima formation have been obtained in rat3 4 5 6 20 29 30 and in guinea pigs.31 However, ACE inhibitors have been found to be ineffective in rabbits,31 pigs,32 baboons,33 and humans,34 suggesting species specificity of ACE treatment. These findings are disappointing in the context that ACE inhibitors are beneficial in human coronary disease and its sequelae.35
In all previous studies, ACE inhibitors were administered either orally,3 5 29 32 intravenously,6 or intraperitoneally as a continuous infusion with an osmotic pump.30 In these cases, systemic effects on hemodynamics cannot be excluded, since they are demonstrated by the marked decrease in blood pressure observed in some cases. In this study, ramiprilat was administered via an Alzet pump in subhypotensive doses (except for the highest dose). Under these conditions, ramiprilat showed a dose-dependent antiproliferative effect. The lower weight gain elicited by the highest ramiprilat dose (3.1 mg · kg-1 · d-1) could be interpreted as an overdose.
Also, ACE not only catalyzes the conversion of Ang I to Ang II but it also degrades BK. The role of BK is mediated at least in part via stimulation of nitric oxide release in endothelial cells, which has antiproliferative effects on vascular smooth muscle cells.36 37 An increased ACE activity could, therefore, contribute to an enhanced degradation of BK and a reduction of nitric oxide production. In fact, Farhy et al29 have suggested that the beneficial effects of ACE inhibitors on neointima formation can be explained by both the stimulation of kinins and NO and the reduction in Ang II production. In this study, we cannot conclude whether neointima formation caused by an upregulation of ACE is mediated through an increased formation of Ang II and/or a decreased disposability of BK. However, the fact that ramiprilat is more potent in inhibiting neointima formation than HR 720 leads to the suggestion that an increase of BK half-life caused by ACE inhibition may play a major role in the protective activity of ramiprilat, as suggested previously by Farhy et al.29
In conclusion, we show that endothelial denudation in the balloon catheter model of the rat induces a local upregulation of ACE before the initiation of neointima formation, which can be prevented by treatment with ramiprilat or HR 720. The exact stimulus that triggers the upregulation of ACE remains to be determined.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a fellowship of the European Community (M.S.F.A.) and a grant from the Deutsche Forschungsgemeinschaft (Pa 332/3-1) (M.P.). The technical help of Heike Marquardt and Daniela Meier is appreciated. We thank Dr Chris E. Talsness for critically reading the manuscript.
Received December 2, 1996; first decision January 2, 1997; accepted January 24, 1997.
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