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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:857-863.)
© 1996 American Heart Association, Inc.

Articles

Angiotensin II-Induced Progression of Neointimal Thickening in the Balloon-Injured Rat Carotid Artery Is AT₁ Receptor Mediated

Ellen M. van Kleef; Jurgen Fingerle; Mat J.A.P. Daemen

the Department of Pathology, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, Netherlands, and the Pharma Division (J.F.), Preclinical Research, F Hoffmann-La Roche Ltd, Basel, Switzerland.

Correspondence to Mat J.A.P. Daemen, Department of Pathology, University of Limburg, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail MDA@ms.azm.2.azm.nl.

	Abstract

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To investigate the relative importance of AT₁ and AT₂ receptors in angiotensin II (Ang II)-induced restimulation of neointimal smooth muscle cell (SMC) DNA synthesis and increased neointimal cross-sectional area (CSA), male Wistar rats were subcutaneously infused for 2 weeks with Ang II and losartan, an AT₁ receptor antagonist, or Ang II and PD123319, an AT₂ receptor antagonist, during the third and fourth week after balloon injury of the left common carotid artery. Concomitantly, all rats received 5-bromo-2'-deoxyuridine to label DNA-synthesizing SMCs. Neointimal CSAs and SMC DNA synthesis were compared with control groups that received Ang II, 0.9% NaCl, losartan, or PD123319. Systolic blood pressure (SBP) was measured at different times during the infusion. Ang II induced an increase in SBP that was significantly different from the SBP in the NaCl group. Infusion of Ang II together with losartan reduced the Ang II-induced increase in SBP to levels comparable with those obtained in the NaCl group. Infusion of Ang II+PD123319 caused an increase in SBP that was comparable with the increase in SBP of the Ang II group and significantly different from the SBP of the NaCl group. Infusion of losartan or PD123319 alone did not affect SBP. Ang II significantly enhanced neointimal CSA (47%, P<.05) compared with the control group infused with NaCl. Losartan significantly reduced Ang II-induced neointimal thickening (neointimal CSA, -37%, P<.05). Infusion of PD123319 together with Ang II did not affect Ang II-induced neointimal thickening. Losartan or PD123319 alone did not reduce neointimal thickening, since the neointimal CSAs in these groups did not differ from the neointimal CSA of the NaCl group. Comparable effects were found for SMC DNA synthesis in the neointima. Ang II infusion increased neointimal SMC DNA synthesis. Addition of losartan reduced the fraction of DNA-synthesizing neointimal SMCs from 23.7±2.1% in the Ang II group to 12.8±1.8% in the Ang II+losartan group, whereas the labeling fraction in the neointima remained 26.6±3.1% in the Ang II+PD123319 group. The labeling fractions in the neointimas of the groups that received losartan or PD123319 alone did not differ from the labeling fraction in the NaCl group. These data indicate that AT₁ but not AT₂ receptors mediate the progression of neointimal thickening induced by delayed application of Ang II in the injured left carotid artery in the rat. Furthermore, these data suggest that AT₁ and AT₂ receptors are not involved in the regulation of normal growth of a neointima in the third and fourth week after balloon injury.

Key Words: restenosis • balloon injury • growth control • AT receptor antagonists

	Introduction

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Although it is well described that Ang II increases balloon catheter-induced neointimal thickening of the rat and rabbit carotid artery,^{1 2 3} it is still not clear which AT receptor subtype is involved in the regulation of neointimal thickening. In vitro, AT₁ receptors appear to mediate the effects of Ang II on SMC DNA synthesis, polyploidy, and protein synthesis,^{4 5 6 7} since these effects can be blocked by administration of the AT₁ receptor antagonist losartan but not the AT₂ receptor antagonist PD123177.^{8 9 10} In vivo, losartan reduces neointimal formation, but the greater effectiveness of angiotensin-converting enzyme inhibitors in the same model might suggest the involvement of AT₂ receptors.¹¹ Indeed, several studies point to a possible involvement of AT₂ receptors in neointimal formation. First, although the presence of AT₂ receptors has been demonstrated in the rat carotid artery media and neointima 15 days after balloon injury,^{12 13 14} controversy exists whether the expression of AT₂ receptors is increased during neointimal thickening after balloon injury.^{13 14} Second, chronic perivascular infusion with the AT₂ receptor antagonist CGP42112A from 6 days before until 14 days after balloon injury of the rat carotid artery reduces SMC proliferation; the authors conclude¹⁵ that AT₂ receptors play a major role in neointimal proliferation. However, because of the high amount of CGP42112A available in the vicinity of the lesion and because of possible partial AT₁ receptor agonistic effects of this drug,¹⁶ CGP42112A may also have affected AT₁ receptors.¹⁷ Moreover, systemic infusions of this compound were not effective.¹⁵ The actual involvement of AT₁ receptors in the formation of a neointima has been shown by many studies in which administration of losartan directly after balloon injury reduced neointimal thickening.^{2 3 11 18 19 20 21 22 23 24} Still, the interpretation of those studies is difficult, since neointimal formation is the result of three sequential processes: medial SMC proliferation, migration of medial SMCs into the intima, and intimal thickening, including both neointimal SMC proliferation and production of extracellular matrix components.²⁵ Since AT₁ receptor antagonists can block medial SMC proliferation and migration,²³ the effects of early administration of these drugs on neointimal formation may be explained by their possible effects on processes in the media rather than the neointima. Moreover, it is evident that factors that control medial SMC growth, such as Ang II, may be rather different from those controlling intimal SMC growth.^{1 26 27} We therefore studied the effects of Ang II and AT receptor antagonists on an already existing neointima by administering the drugs during the third and fourth weeks after balloon injury. During this period neointimal mass continues to increase,²⁵ and administration of Ang II restimulates neointimal SMC DNA synthesis and enhances neointimal CSA.¹ Since all known Ang II-mediated effects on SMCs appear to be mediated via AT₁ receptors,¹⁷ we hypothesized that AT₁ receptors are also involved in Ang II-induced neointimal SMC DNA synthesis and neointimal thickening after balloon injury.

We investigated the relative importance of AT₁ receptors and AT₂ receptors in nonstimulated SMCs and Ang II-induced restimulation of neointimal SMC DNA synthesis and increased neointimal CSA by investigating the effects of delayed administration of Ang II and the specific AT₁ (losartan) and AT₂ (PD123319) receptor antagonists. The results indicate that AT₁ but not AT₂ receptors are involved in the Ang II-induced neointimal SMC DNA synthesis and increased neointimal CSA in the injured rat carotid artery during the third and fourth weeks after injury. Since infusions of losartan or PD123319 alone had no effect on neointimal CSA and DNA synthesis, AT₁ and AT₂ receptors seem not to be involved in the growth of the neointima in the third and fourth weeks after balloon injury.

	Methods

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Male adult Wistar rats (body weight, 350 to 420 g) obtained from Biological Research Laboratories, RCC, were provided food and tap water ad libitum. Experiments were performed in accordance with institutional guidelines. At day 0 the left common carotid artery of all rats was balloon injured. Rats were divided into six groups, and infusions of either Ang II (n=8), 0.9% NaCl (n=7), Ang II and losartan (n=8), Ang II and PD123319 (n=8), losartan (n=6), or PD123319 (n=6) were administered during the third and fourth weeks after balloon injury. At the same time BrdU was infused to label DNA-synthesizing SMCs.

BP Measurement
SBP was measured by tail-cuff plethysmography (blood pressure recorder 8005, W&W Electronics) on conscious restrained rats. SBP was measured 5 days before ballooning (day 0), in the second week after balloon injury (days 8 and 10), and during infusion of the drugs (days 17, 22, and 24). Rats were coded so that all analyses were performed with the investigators blinded to the treatment groups.

Balloon Injury of the Left Common Carotid Artery
The rats were anesthetized by intraperitoneal administration of sodium pentobarbital 60 mg/mL (Veterinary Laboratories Inc). A water-filled embolectomy catheter (2F Fogarty, Edwards Laboratories) was inserted into the left external carotid artery and passed through the common carotid artery into the descending thoracic aorta. The balloon was inflated to obtain slight resistance and rotated while it was withdrawn. After three repeated denudations the catheter was removed, the external carotid was ligated, and the wound was closed. Blood flow was maintained through the common and internal carotid arteries.²⁸

Drug Infusion
Drug infusion was started 2 weeks after balloon injury. Drugs were administered by using osmotic minipumps (Alzet model 2002, Alza Corp) that were implanted subcutaneously between the shoulder blades under 1% halothane anesthesia (by inhalation, Pitman-Moore). [Val⁵]-Ang II (Sigma Chemical Co) was dissolved in 0.9% NaCl and infused at a rate of 0.5 mg·kg^-1·d ^-1. Losartan (kindly provided by Du Pont) was infused at a rate of 15 mg·kg^-1·d ^-1; PD123319 (kindly provided by Parke-Davis) was infused at a rate of 3 mg·kg^-1·d ^-1. At the same time, all rats received BrdU (Serva) in 0.9% NaCl at an infusion rate of 0.8 mg·kg^-1·d^-1 via a separate subcutaneous osmotic minipump (Alzet model 2002).

Tissue Processing
At the end of week 4, the rats were killed by an overdose of anesthetic and perfusion-fixed with 4% phosphate-buffered paraformaldehyde via a cannula inserted into the left ventricle of the heart (perfusion pressure, 100 mm Hg). Both the left and right carotid arteries were isolated. At 1 cm from the aortic arch, a 2-mm ring of each carotid artery was cut and routinely processed for paraffin embedding. This section represents the central third part of the carotid artery where the artery remains devoid of endothelium.²⁸ Since all carotid segments were cut from the same site, diversity in size as a result of other effects (such as the presence of endothelium) was reduced to a minimum. Cross sections (4 µm) were cut and stained with Lawson's solution (Boom BV) to measure the CSAs of the media and neointima by using a computerized morphometry system (Quantimet 570, Leica). The CSA of the media was defined as the area enclosed within the external and internal elastic lamina; the CSA of the neointima, by the area enclosed within the internal elastic lamina and the lumen. Two adjacent sections per carotid artery were evaluated. This protocol was followed by two independent observers, whereby the CSAs for each animal were evaluated by measuring each section twice. The intraobserver and interobserver variations were <5%.

DNA Synthesis
BrdU-labeled nuclei were visualized by using an indirect immunohistochemical technique.²⁹ After rehydration of 4-µm cross sections, blocking of endogenous peroxidase, washing in phosphate-buffered saline (pH 7.4), and digestion in 0.05 mg/mL pepsin (Boehringer) in 0.1N HCl for 30 minutes at 37°C, the sections were incubated in 2N HCl for 15 minutes at 37°C and washed in 0.1 mol/L sodium tetraborate (pH 8.5). Sections were incubated with a monoclonal anti-BrdU antibody (Eurodiagnostics) for 1 hour at 37°C followed by incubation with biotinylated rabbit anti-mouse IgG (Amersham) at room temperature for 30 minutes. Sections were labeled for 30 minutes with an avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Labs). 3,3' Diaminobenzidine was used as the chromogen, and the sections were counterstained with hematoxylin. Omission of the primary antibody and staining with mouse IgG served as negative controls. After immunostaining, the cumulative labeling fraction ([BrdU-positive cells/total number of cells]x100%) was determined by one observer who counted the number of labeled cells and the total number of cells in the media or neointima.

Dose-Finding Studies With Losartan and PD123319
We have shown³⁰ that losartan 15 mg·kg^-1·d^-1 results in a 15- to 30-fold shift of the pressor response curve to Ang II. Early administration of this dose is also effective in inhibiting neointimal formation in the rat.^{18 23 24}

The dose of PD123319 used in the present study decreases interstitial DNA synthesis in the rat heart after myocardial infarction, whereas AT₁ receptor blockade has no effect in that model.^{30 31} The dose of PD123319 was based on concentration measurements in plasma after a 1-hour infusion in rats. Infusion of 100 µg·kg^-1·min^-1 results in plasma levels of 10 µmol/L (M. DeGasparo, unpublished data, 1995). From this we extrapolated that infusion of 3 mg·kg^-1·d ^-1 (2 µg·kg^-1·min^-1) would result in a plasma concentration on the order of 200 nmol/L. Given the IC₅₀ (17 nmol/L) of PD123319 in the human uterus, this suggested that infusion of 3 mg·kg^-1·d^-1 would result in a plasma concentration on the order of 10 times the IC₅₀ for AT₂ receptors. In rats, the IC₅₀ of PD123319 for the vascular smooth muscle AT₁ receptor is >10 µmol/L, which should leave this receptor unaffected at the dose used.

The latter was verified in separate groups of rats that were subcutaneously infused with saline (n=6) or PD123319 3 mg·kg^-1·d^-1 (n=6) for 2 weeks. At the end of the infusion period rats were anesthetized with pentobarbital 60 mg/kg IP, and catheters were inserted into the right femoral vein for injections and into the right femoral artery for measurement of BP. Rats were placed on a heating pad (37°C); following establishment of baseline BP, 0.03 µg/kg Ang II was injected. In saline-infused rats, the resultant increase in mean arterial pressure was 36±3 mm Hg (mean±SEM); in the PD123319-infused rats, this dose of Ang II increased mean arterial pressure by 37±3 mm Hg, suggesting a total lack of inhibition of the pressor response to Ang II, which we interpret as a lack of AT₁ receptor antagonism.

Neointimal CSA 2 Weeks After Balloon Injury
The left carotid artery of an additional group of six male Wistar rats of comparable body weight was balloon injured according to the protocol described above. After 2 weeks the rats were killed and perfusion-fixed with 4% phosphate-buffered paraformaldehyde, and tissue processing and measurement of intimal and medial CSAs were performed as described above.

Statistics
Data are expressed as mean±SEM. SBPs were analyzed by two-way ANOVA and CSAs by one-way ANOVA (SuperAnova, Abacus Concepts). In the case of overall significance (P<.05), multiple comparisons were made between different means (t test). The level of significance was adjusted by Bonferroni t test. In the case of BrdU indices, values were normalized by logarithmic transformation before subjection to ANOVA. Instead of t tests, contrasts of selected pairs of group means were computed and adjusted by using Bonferroni-Holm corrections.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Ang II and AT Receptor Antagonists on SBP
At the start of the experiment, basal SBPs were approximately 145 mm Hg. Balloon injury (day 0) did not affect SBP. Drugs were infused for 2 weeks, starting at day 14. Infusion of Ang II increased SBP by approximately 35 mm Hg over the time of infusion, which differed significantly from the NaCl group (Fig 1). Infusion of Ang II together with losartan reduced the Ang II-induced increase in SBP, which was comparable with that obtained in the NaCl group. Infusion of Ang II+PD123319 caused an increase in SBP that was comparable with the increase in SBP of the Ang II group and significantly different from the SBPs of the NaCl group. Infusion of losartan alone did not lower SBP in comparison with the NaCl group. Infusion of PD123319 had no effect on SBP (Fig 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Line graph. At day 0 balloon injury (BI) of the left carotid artery was performed. Two weeks later, infusions (I) were started with Ang II and AT receptor antagonists for an additional 2 weeks. Rats were randomly assigned to different groups. Four weeks after balloon injury rats were killed (S). Data are mean±SEM. P<.05 vs control (NaCl group) over time.

Effects of Ang II and AT Receptor Antagonists on Medial and Neointimal CSAs
Effects on Injured (Left) Carotid Neointima
Four weeks after balloon injury the neointimal CSA was 0.17±0.02 mm² in the group treated with 0.9% NaCl (Figs 2 and 3). Infusion of Ang II increased the neointimal CSA by 47%. Addition of losartan to Ang II reduced the neointimal CSA to NaCl group levels, but infusion of Ang II and PD123319 resulted in a neointimal CSA that was comparable with the neointimal CSA of the Ang II group. The neointimal CSAs did not differ between the losartan and NaCl groups, but the neointimal CSA of the PD123319 group was reduced by 30% compared with the NaCl group (NS).

View larger version (702K): [in this window] [in a new window]   Figure 2. Photomicrographs showing Lawson-stained cross sections of injured rat carotid artery 28 days after balloon injury treated with (A) Ang II, (B) Ang II+losartan, (C) Ang II+PD123319, (D) NaCl, (E) losartan, and (F) PD123319.

View larger version (28K): [in this window] [in a new window]   Figure 3. Bar graph shows effects of Ang II and AT receptor antagonists on neointimal CSAs of carotid arteries 28 days after balloon injury. Data are expressed as percentages of the neointimal CSA of the NaCl group, which was set at 100%. *P<.05 vs control (NaCl group).

Effects on Injured (Left) Carotid Tunica Media
Ang II infusion showed a nonsignificant tendency to increase medial CSA in comparison with the NaCl group. Addition of losartan to Ang II significantly reduced medial CSA by -20% compared with the Ang II group (Table 1), whereas addition of PD123319 had no effect. Infusion of losartan or PD123319 alone resulted in medial CSAs that did not differ from the medial CSA of the NaCl group.

View this table: [in this window] [in a new window]   Table 1. Effects of Ang II and AT Receptor Antagonists on Medial Cross-sectional Areas of the Balloon-Injured Left Carotid and Noninjured Right Carotid Artery Tunica Media

Effects on Noninjured (Right) Carotid Tunica Media
Infusion of Ang II had no significant effect on the medial CSA of the noninjured carotid artery (Table 1). Infusion of losartan together with Ang II resulted in a reduction in medial CSA in comparison with the Ang II group, whereas addition of PD123319 to Ang II had no effect on medial CSA. Treatment with losartan alone resulted in a smaller medial CSA than the medial CSA of the groups treated with Ang II alone or NaCl.

Effects of Ang II and AT Receptor Antagonists on SMC DNA Synthesis
Infusion of Ang II caused a threefold increase in neointimal SMC DNA synthesis compared with the NaCl group (Fig 4). Addition of losartan reduced the Ang II-induced increase in BrdU labeling percentage to control (NaCl) levels, whereas the BrdU labeling index in the Ang II+PD123319 group remained elevated. Infusion of losartan or PD123319 alone had no effect on SMC DNA synthesis.

View larger version (92K): [in this window] [in a new window]   Figure 4. Photomicrographs of cross sections of balloon-injured left carotid arteries 28 days after balloon injury stained immunohistochemically for anti-BrdU and treated with (left) NaCl and (right) Ang II (both, x400).

The effects on the BrdU labeling index in the medial SMCs of both the injured left and noninjured right carotid arteries were comparable with those found in the neointima (Table 2). Ang II increased the BrdU labeling fraction in the left carotid media from 0.2±0.2% in the NaCl group to 2.4±1.1% in the Ang II group, and in the right carotid media from 0% to 1.2±0.1%. Coinfusion with losartan reduced the labeling fraction in both carotid medias, whereas coinfusion with PD123319 had no effect. Infusion of losartan or PD123319 alone had no effect on the BrdU labeling fraction.

View this table: [in this window] [in a new window]   Table 2. Effects of Ang II and AT Receptor Antagonists on BrdU Labeling Fraction in Balloon-Injured Left Carotid and Noninjured Right Carotid Arteries

Neointimal CSA 2 Weeks After Balloon Injury
To determine the amount of neointimal growth during the third and fourth weeks, the carotid neointimal CSA of the rats killed at 2 weeks was compared with the carotid neointimal CSA of the NaCl group killed 4 weeks after balloon injury. At 2 weeks after injury, the mean carotid neointimal CSA was 0.10±0.01 mm². The neointimal CSA of the NaCl group at 4 weeks after balloon injury was 0.l7±0.02 mm², which implies a 41% increase in neointimal area during the third and fourth weeks after balloon injury.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study focuses on the effects of Ang II on neointimal SMCs, which play a key role in atherogenesis and restenosis. We modified the classic rat carotid balloon-injury model in such a way that the interventions were not started immediately after balloon injury but 2 weeks later. At that time a neointima has already been formed, and neointimal growth will be the result of neointimal SMC growth and not medial SMC growth or migration, which occur only during the first week after injury.²⁷ In the third and fourth weeks after injury the intima is, however, still responsive to growth stimuli, such as Ang II.¹ Our modified balloon-injury model, the "restimulation model," may thus better represent human atherogenesis and/or restenosis. The present study indicates that AT₁ but not AT₂ receptors mediate the increased neointimal thickening caused by delayed application of Ang II in the injured left carotid artery in the rat. Furthermore, these data suggest that neither AT₁ nor AT₂ receptors are involved in the regulation of normal growth of a neointima in the third and fourth week after balloon injury, since neither losartan nor PD123319 reduced neointimal SMC DNA synthesis or neointimal CSA in comparison with NaCl-treated control rats.

Although the present data clearly indicate that AT₁ receptors are involved in the stimulation of Ang II-induced neointimal thickening and SMC DNA synthesis, it is not clear whether this is due to direct or indirect effects of Ang II. Direct effects suggest that the effects of Ang II on neointimal thickening and DNA synthesis are mediated by AT₁ receptors located on the vascular smooth muscle, whereas indirect pathways may include, among others, an increased BP as a result of Ang II infusion and an increased activity of the sympathetic nervous system by Ang II (Fig 5). Increased activity of the sympathetic nervous system is facilitated by stimulation of peripheral adrenergic function³² through AT₁ receptors present on the adrenergic nerve terminals,³³ resulting in increased release of noradrenaline^{34 35} and subsequent stimulation of ₁-adrenoreceptors (Fig 5). The involvement of the sympathetic nervous system and ₁-adrenoreceptors in Ang II-mediated vascular growth has been demonstrated for medial SMC DNA synthesis, since continuous infusion of Ang II together with the ₁-adrenoreceptor blocker prazosin reduces medial SMC DNA synthesis in the uninjured rat carotid artery and thoracic aorta.²⁹ However, infusion of Ang II together with the ₁-adrenoreceptor blocker doxazosin during the third and fourth week after balloon injury of the rat carotid artery reduces SMC DNA synthesis in the media but not in the neointima.^{29 36} In a comparable balloon-injury experiment, the ₁-adrenoreceptor agonist phenylephrine stimulated medial but not neointimal SMC DNA synthesis.³⁷ Thus, the data from these studies indicate that at least during the third and fourth week after injury, Ang II-induced neointimal SMC proliferation is not mediated by ₁-adrenoreceptors.

View larger version (10K): [in this window] [in a new window]   Figure 5. Schematic representation of possible direct and indirect pathways via which Ang II could exert its effects on neointimal SMC DNA synthesis and neointimal CSA. "Direct" represents a direct action of Ang II on vascular AT receptors; "indirect," an indirect action of Ang II whereby Ang II exerts effects on neointimal SMCs via increased BP and increased sympathetic activity (see "Discussion"). NE indicates norepinephrine.

A second indirect pathway involves increased BP as a result of Ang II (Fig 5). From the studies presented here a mediatory role of BP cannot be excluded, since infusion of Ang II and losartan resulted in both a reduction in BP as well as a reduction in neointimal CSA and SMC DNA synthesis. However, several other studies show that BP increases are not a prerequisite for Ang II-mediated vascular hypertrophy. For instance, infusion of both Ang II and hydralazine in rats reduces the Ang II-induced BP increase but not the Ang II-induced increase in medial hypertrophy of the rat superior mesenteric artery.³⁸ Also, whereas coinfusion of Ang II and the ₁-adrenoreceptor blocker prazosin has no effect on the increase in BP caused by Ang II, the Ang II-stimulated medial SMC DNA synthesis is reduced.²⁹ In a recent study the possible interaction between the renin-angiotensin system, vascular hypertrophy, and BP was addressed more directly by in vivo transfer of the human angiotensin-converting enzyme gene into rat carotid arteries.³⁹ The increased vascular angiotensin-converting enzyme expression resulted in vascular hypertrophy without an increase in circulating angiotensin or BP.³⁹ Thus, it is not likely that BP plays a key role in Ang II-induced effects on vascular growth.

This leaves the possibility that the effects of Ang II on neointimal thickening and DNA synthesis are mediated by AT₁ receptors located on vascular SMCs (Fig 5). Support for possible direct effects comes from in vitro studies in which Ang II stimulates growth via AT₁ receptors in a number of cell types, including SMCs and cardiac myocytes.^{8 40 41}

The results of the present study indicate that AT₂ receptors are not involved in the Ang II-induced neointimal thickening and SMC DNA synthesis. Our dose-finding studies indicate that the dose of PD123319 that was used did not affect AT₁ receptors, since the pressor response curve to Ang II was not shifted to the right in the PD123319-treated rats. The compound also had no effects on the Ang II-induced BP increase after balloon injury (Fig 1). Furthermore, preliminary data from another study show that this dose of PD123319 reduces interstitial DNA synthesis after rat myocardial infarction,³¹ indicating that at this dose PD123319 can have biological effects.

A second finding in this study was that administration of losartan or PD123319 alone did not affect neointimal growth during the third and fourth weeks after balloon injury. This suggests that neointimal growth during this period, which is mainly due to synthesis and accumulation of extracellular matrix components,²⁵ is not mediated by AT receptors or that the relative increase in neointimal growth is too small during the third and fourth weeks to detect an effect of AT receptor antagonists alone. The latter possibility seems unlikely, since the increase in neointimal CSA during the third and fourth weeks still comprises 41% of the total neointimal CSA.

An understanding of the growth regulation of the intima in experimental animal models is useful for a better understanding of human intimal lesions. Experimental models focusing on medial SMC growth regulation may give inadequate results since the regulation of neointimal SMC growth differs from that of medial SMC growth.^{27 42 43 44 45} This study further shows that the growth regulation of a restimulated intima may differ from that of normal intimal growth.

In conclusion, AT₁ but not AT₂ receptors are involved in Ang II-induced progression of neointimal thickening and SMC DNA synthesis in the third and fourth weeks after balloon injury of the rat carotid artery. The data further suggest that AT₁ and AT₂ receptors do not play a prominent role in the regulation of normal neointimal growth during this time.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II BP = blood pressure BrdU = 5-bromo-2'-deoxyuridine CSA = cross-sectional area SBP = systolic blood pressure SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by the Netherlands Heart Association, grant NHS93165. The authors wish to thank Petra Aarts, Feridun Shodjai, Georges Wdonwieki, Phillipe Wyss, and Edwin Weijers for excellent technical assistance.

Received March 31, 1995; revision received January 26, 1996;

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