© 1996 American Heart Association, Inc.
Articles |
Smooth Muscle DNA Replication in Response to Angiotensin II Is Regulated Differently in the Neointima and Media at Different Times After Balloon Injury in the Rat Carotid Artery
Role of AT1 Receptor Expression
the Department of Pathology (D. deB., J.E.S., S.M.S.) and Surgery (A.W.C.), University of Washington, Seattle, and the Section on Pharmacology (M.V., J.M.S.), Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md.
Correspondence to Dr D. deBlois, Centre de Recherche Hotel-Dieu de Montreal, 3840 St Urbain St, Montreal, Quebec H2W 1T8, Canada. E-mail debloisd@ere.umontreal.ca.
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Abstract |
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We have reported that angiotensin II (Ang II) infusion to rats during the third and fourth weeks after vascular injury stimulates DNA replication in a larger proportion of smooth muscle cells (SMCs) in the arterial neointima than in the underlying media or the normal arterial media. Whether this increased responsiveness to Ang II is a transient or stable property of neointimal cells after vascular injury remained unclear. The present study examined smooth muscle DNA replication in response to Ang II infusion (250 ng·kg-1·min-1 for 2 weeks) at 3 to 4, 9 to 10, or 27 to 28 weeks after balloon injury to the rat carotid artery. Control rats received Ringer's lactate. BrdU (0.8 mg·kg-1·d-1) was coinfused to label replicating DNA. The increased replicative response to Ang II in the neointima versus the normal arterial media did not persist beyond the period of rapid lesion growth shortly after injury, even in neointimal areas without endothelial regeneration. By 9 to 10 weeks after injury, replication frequencies were comparable in the neointima and the normal arterial wall. In the presence of a regenerated endothelium, neointimal DNA replication was lowered but not abolished. After the early period, however, the most marked difference may be the loss of ability of medial SMCs to respond mitogenically to systemic Ang II. As a consequence, Ang II–induced DNA replication in injured arteries was greater in the neointima than in the underlying media at all times studied after injury. DNA replication levels correlated with AT1 receptor levels in the injured artery neointima but not media, as shown by receptor binding in vascular sections at 3 and 10 weeks after injury. The growth response to systemic Ang II is differentially regulated in adjacent smooth muscle layers in the injured arterial wall in vivo via mechanisms that include, but are not restricted to, the regulation of AT1 receptor expression in SMCs.
Key Words: vascular smooth muscle • DNA replication • angiotensin II receptors • neointima
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Introduction |
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The rat carotid artery injured with a balloon catheter is a widely used model for the study of factors controlling intimal lesion formation after endothelial injury. In this model, basic fibroblast growth factor is a key mitogen for SMCs in the media but not the neointima.1 2 3 Platelet-derived growth factor4 5 6 and transforming growth factor-ß7 8 are endogenous stimulants of neointimal formation, though not mainly via an effect on SMC proliferation in the injured wall. Animal studies with inhibitors of the renin-angiotensin system have shown that Ang II stimulates intimal lesion growth in arteries during atherosclerosis9 10 11 12 13 or after balloon catheterization.14 15 16 17 18 19 Consistent with these observations, Ang II stimulates DNA replication in arterial SMCs in vivo.20 Moreover, infusion of Ang II to rats during the third and fourth weeks after arterial injury stimulates DNA synthesis more markedly in SMCs in the neointima than in the underlying media or the normal arterial wall. It has remained unclear, however, whether the enhanced replicative response to Ang II is a transient phenomenon associated with the early phase of vascular wound healing or, alternatively, a stable characteristic of neointimal SMCs in vivo. Recently, AT1 receptors for Ang II have been shown to be overexpressed in the developing neointima after vascular injury.21 22 Together, these data suggest an association between vascular AT1 receptor expression and the replicative response to Ang II.
The present study was designed to further characterize SMC growth during Ang II–induced hypertension and its possible correlation with AT1 receptor levels as a function of time after formation of a neointima in the balloon-injured carotid artery of the rat. The growth response of SMCs to Ang II and the expression of Ang II receptors were examined in the arterial media and neointima. We examined intimal growth in areas chronically lacking ECs in vivo as well as in areas overlaid with a regenerated endothelium. The results reveal prolonged differences in the growth response to systemic Ang II in vivo between the medial and intimal SM layers of the injured arterial wall.
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Methods |
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Vascular Injury
A 2F balloon embolectomy catheter (V. Mueller) was used to cause vascular injury to the left common carotid artery of 48 male Sprague-Dawley rats (Zivic Miller; 400 to 450 g) previously anesthetized with a single injection of ketamine (80 mg/kg IP) and xylazine (4 mg/kg IP). The balloon catheter was inserted into the left common carotid artery via the external carotid artery, passed to the level of the aortic arch, inflated, and withdrawn with a twisting motion to deendothelialize and dilate the whole length of the common carotid artery. This procedure was performed a total of three times before the external branch of the carotid artery was ligated and the neck incision closed. The right carotid artery was not manipulated. All surgical procedures were conducted in accordance with institutional guidelines.
Drug Infusion
To study SMC growth regulation by Ang II as a function of time after neointimal formation, rats were given a 2-week infusion of Ang II (250 ng·kg-1·min-1 SC) or its Ringer's lactate vehicle starting at 2, 8, or 26 weeks after balloon injury to the left carotid artery. Rats were randomly assigned to a specific schedule of drug treatment at the time of arterial injury, and at the beginning of drug treatment rats were again randomly assigned to receive Ang II or Ringer's lactate. Drugs were infused continuously via osmotic pumps (Alzet model 2002, Alza Corp) implanted subcutaneously in the back under general anesthesia as described above. All rats were also implanted with a second osmotic pump that delivered a continuous infusion of the nonradioactive thymidine analogue BrdU (0.8 mg·kg-1·d-1), which is incorporated into DNA during replication in vivo.23 Ang II and BrdU were from Sigma Chemical Co.
BP and Body Weight
Systolic BP was measured in all rats by using tail-cuff plethysmography (Narco Biosystems). Measurements were started 1 week before treatment and performed on conscious, restrained rats that had been trained for the procedure. Rats were weighed before, during, and at the end of the 2-week drug infusion.
Immunohistochemistry
To select denuded arterial segments, Evans blue was given intravenously to all rats 10 minutes before they were killed by an overdose of pentobarbital 25 mg/kg IP. Both common carotid arteries were excised and cleaned of adherent connective tissue before the segments were cut and immersion-fixed in 4% paraformaldehyde. Specimens were processed according to routine histological procedures and embedded in paraffin. Tissue sections (5 µm) were obtained for immunohistochemistry.
Incorporation of BrdU in the nucleus of SMCs was examined by using an indirect peroxidase-labeled antibody technique.24 Briefly, deparaffinized tissue sections were treated with H2O2 (0.3% in methanol) for 20 minutes to block endogenous peroxidase, washed in 0.05 mol/L Tris-HCl (pH 7.6), digested with 0.2 mg/mL pepsin (Boehringer Mannheim Biochemicals) in 0.1N HCl for 30 minutes at 37°C to expose the DNA, incubated in 1.5N HCl for 30 minutes at 37°C to denature the DNA, and washed in 0.1 mol/L sodium tetraborate (pH 8.5) to fix the DNA. Incubation of sections for 1 hour at 37°C with a monoclonal mouse anti-BrdU IgG (Euro-Diagnostic BV) was followed by incubation with biotinylated horse anti-mouse IgG (rat adsorbed; Boehringer Mannheim Biochemicals) for 45 minutes at 22°C. An avidin-biotin complex (ABC Elite, Vector) conjugated to horseradish peroxidase was applied for 30 minutes at 22°C. The chromogen 3,3'-diaminobenzidine, which precipitates as a dark crystal within 10 minutes in the presence of peroxidase activity, was used to stain the immunoreactive nuclei. Sections were counterstained with hematoxylin before being permanently mounted under coverslips. The number of SMCs in the media and neointima per cross section was evaluated by counting under light microscopy. Results are expressed separately for neointima and media as the cumulative BrdU-labeling fraction, ie, the percentage of nuclei having synthesized DNA at any given time during the 2-week period of subcutaneous drug administration or as the absolute number of labeled nuclei.
In injured arteries isolated at 10 weeks or later after injury, a segment with regenerated endothelium was examined in addition to the segment lacking endothelial regrowth. The presence of endothelium overlying the neointima was confirmed by immunohistological detection of the EC marker von Willebrand factor by using a rabbit anti-human factor VIII–related antigen polyclonal antibody (Dako Laboratory) in tissue cross sections according to a procedure similar to the BrdU procedure described above, with the omission of the 1.5N HCl and 0.1 mol/L sodium tetraborate incubation steps. Neointima that has been reendothelialized lacks a clear boundary, such as the internal elastic lamina in uninjured carotid arteries, between ECs and SMCs. Thus, to exclude the ECs overlying the neointima from the calculations for the SMC BrdU-labeling fraction, the nuclei situated at the lumenal edge of the reendothelialized neointima were not counted.
Measurement of Vascular Cross-sectional Area
Two nonconsecutive cross sections (5 µm) of each artery were stained with the elastin fiber–specific stain orcein, and cross-sectional areas were measured by using light videomicroscopy with a computerized morphometry system (BioScan). Medial area was defined as the area enclosed between the external and internal elastic laminae, and intimal area was defined as the area between the internal elastic lamina and the lumen perimeter.
Angiotensin Receptor-Binding Assay
Segments of carotid arteries were frozen immediately in optimal cutting temperature medium (Miles) after isolation at 3 or 10 weeks after injury from Ringer's-infused animals (n=5 and n=11, respectively). Only segments without endothelium were examined in injured arteries. For each animal the contralateral uninjured artery was processed along with the injured artery. Vascular sections (16 µm) were obtained and processed for quantitative autoradiography by using the radiolabeled tracer [125I]Sar1 Ang II (Peninsula; iodinated by New England Nuclear; specific activity, 2200 Ci/mmol) at saturating (3 nmol/L) and half-maximal (0.5 nmol/L) concentrations and unlabeled losartan (10 µmol/L) and CGP 42112 (0.1 µmol/L) as competitors for the AT1 and AT2 receptors, respectively.22 Bound radioactivity was quantified by exposing sections and 125I-labeled Micro-scale standards to Hyperfilm-3H (both, Amersham Corp), measuring the optical density in the developed films, and generating a standard curve for disintegrations per minute per milligram of protein.25 In uninjured arteries, binding was determined in the media plus endothelium; in injured arteries, binding was evaluated separately for the neointima and the underlying media. To identify areas corresponding to the neointima and the media in the autoradiograms of the injured arteries, tissue sections were counterstained with hematoxylin-eosin, and the light-transmission micrograph for each specimen was projected on a computer screen side-by-side with the corresponding image of the autoradiogram by using image-analysis software (Image, NIH). Binding data are expressed in femtomoles per milligram of protein.
Statistical Analysis
Results are expressed as mean±SEM. Student's t test was used to compare the data between the groups treated with Ang II and Ringer's lactate. Two-way ANOVA followed by Sheffe's F test were used to conduct multiple comparisons. A probability of less than .05 for wrongfully rejecting the null hypothesis was considered significant.
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Results |
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Systemic Changes During Ang II Infusion
Systolic BP values in rats before drug treatment were similar for all experimental groups of animals (133±4 mm Hg; n=48). Continuous infusion of Ang II (250 ng·kg-1·min-1) caused a progressive and significant increase in systolic BP, up to 179±8 mm Hg by 1 week and 195±5 mm Hg by 2 weeks. In addition, while all rats gained weight with time after vascular injury, there was a significant reduction (10% to 15%) in final body weight as a result of Ang II infusion.
DNA Replication in Uninjured Arteries
As we have shown,20 infusion of Ang II significantly increased DNA synthesis in the media of uninjured carotid arteries (Table 1
). This effect did not differ among the three groups of rats infused with the peptide at various times after vascular injury (P=.2 by ANOVA). The number of nuclear profiles in the media of the normal carotid artery showed no significant change (P=.2 by ANOVA) with time or treatment (Ang II group=353±13 and Ringer's group=376±13; n=48).
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DNA Replication in Previously Injured Arteries
The mitogenic effect of Ang II was significant in the media of the injured carotid artery at 3 to 4 weeks after injury, although the response was less pronounced than in the media of the contralateral carotid artery without injury (Table 1
). By 9 to 10 weeks after injury, however, Ang II infusion failed to stimulate DNA synthesis in the media of the injured carotid artery.
In contrast to SMCs in the injured media, SMCs in the neointima remained consistently responsive to Ang II as a mitogen as late as 6 months after vascular injury (Table 1). At all intervals, SMCs undergoing DNA replication in the neointima were mainly situated near the lumenal edge of the lesion, although replicating cells were also observed in the deeper layers of the lesion (Fig 1). The number of nuclear profiles in the injured artery media or neointima was not significantly changed with treatment (eg, at 4 weeks, there were 408±30 and 1654±149 profiles, respectively, with Ang II versus 390±20 and 1634±183 profiles, respectively, with Ringer's lactate).
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Analysis of Ang II proliferative effects in injured versus uninjured arteries was complicated by the very large differences in the baseline rates of DNA replication; eg, in Ringer's lactate–treated specimens at 3 to 4 weeks, the BrdU-labeling fraction was 14.0±1.9% in the neointima versus 2.3±0.7% in the uninjured arterial media. To determine statistically whether Ang II elicited an enhanced proliferative response at 3 to 4 weeks after injury, the difference between the labeling fractions in the neointima and contralateral normal arterial media was calculated individually for each animal. Thus, at 3 to 4 weeks after injury, the neointima–normal media difference in labeling fraction was 12.4±2.0% in rats infused with Ringer's, whereas this difference increased to 22.0±4.3% in rats infused with Ang II (P<.05). Infusion of Ang II beyond the second month after injury, however, failed to elicit replication in a larger fraction of SMCs in the neointima than the normal arterial wall. Thus, at the later times the enhanced response in the neointima over the underlying media during Ang II infusion may mostly reflect a loss of mitogenic effect in the media.
Ang II Receptor Binding
We investigated the expression of Ang II receptors in the carotid artery (Fig 2) at times after balloon injury, when the proliferative response to Ang II is altered. The level of AT1 receptor binding in the neointima at 3 weeks was significantly higher than that in the underlying media, the normal arterial wall, or the neointima and injured media at 10 weeks after injury. AT1 binding was predominant close to the lumenal surface of the neointima at 3 weeks22 ; at 10 weeks, binding in the neointima was low and showed no clear increase near the lumen (not shown). At both times after injury, the relative order of AT1 binding was neointima>injured arterial media
normal arterial media. The injured arterial media and the normal arterial wall showed no significant difference in AT1 binding at either 3 or 10 weeks. Results were similar at saturating (3 nmol/L) or half-maximal (0.5 nmol/L) concentrations21 22 of tracer, suggesting that AT1 receptor affinity was not changed in the different SM layers at the different times after injury. No AT2 binding activity was detected in the normal or injured carotid artery.21 22
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Effect of Endothelium on SM DNA Replication
Because there have been many claims that reendothelialization inhibits neointimal SMC proliferation after vascular injury,26 27 28 we asked whether the presence or absence of endothelium affected the SMC replicative response to Ang II in the neointima. We took advantage of the fact that endothelial regeneration from the extremities of the balloon-denuded carotid artery typically stops before reendothelialization of the neointima is completed. In the present study, the middle segment (8 to 10 mm) of the injured carotid artery lacked an endothelium as late as 6 months after balloon denudation.26 27 28 This was confirmed in all injured arteries by Evans blue exclusion and by immunohistochemistry for von Willebrand factor, an EC marker in arterial cross sections (not shown). The presence of an endothelium over the neointima reduced but did not abolish the replicative response to Ang II in the neointima at 9 to 10 or 27 to 28 weeks after injury (Table 2). No data is available at 3 to 4 weeks because reendothelialization of the neointima was inconsistent at that early time after denudation.
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Interestingly, the neointima with endothelium and the normal arterial media (also with endothelium) showed comparable values in absolute cell growth, ie, in total number of BrdU-labeled cells, in control as well as Ang II rats, even though the percentage of labeled cells was markedly lower in the neointima with endothelium. This reflects the higher total cell number in the neointima versus the normal arterial media. In addition, the absolute cell growth was significantly greater in neointimal areas without endothelium versus neointima with endothelium. For instance, in control rats at 27 to 28 weeks after injury the total number of BrdU-labeled nuclei was 9±5 in the normal arterial media, 4±1 in the neointima with endothelium, and 25±11 in the neointima without endothelium. With Ang II, the total number of BrdU-labeled nuclei was significantly increased to 43±12 in the normal arterial media, 20±8 in the neointima with endothelium, and 104±11 in the neointima without endothelium.
Vascular Hypertrophy During Ang II Infusion
Table 3 shows the effect of the Ang II infusions at various times after balloon injury on the cross-sectional area of the normal and injured carotid arteries. Ang II caused significant increases in the mass of the uninjured carotid artery in all groups of rats. In contrast with the normal arterial wall, the injured artery showed no significant change in mass with Ang II at any of the times studied after vascular injury. Examination of data from all three groups of animals infused with Ringer's lactate suggests that the medial cross-sectional area of the uninjured carotid artery may increase with age.
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Discussion |
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The present data extend our previous observations by showing that SMCs in the neointima overexpress AT1 receptors and exhibit an enhanced ability to undergo DNA replication in response to Ang II infusion in vivo, but only transiently during the early phase of neointimal formation after vascular injury. As early as 2 months after injury, the neointima and normal arterial wall showed comparable rates of DNA replication in control as well as Ang II rats. Moreover, the neointimal levels of AT1 receptor at 3 and 10 weeks reflected the BrdU-labeling fraction in the neointima during the corresponding periods. Responsiveness to Ang II as a mitogen was completely lost in the media by 9 to 10 weeks after injury, whereas Ang II remained a significant stimulant for cells in the neointima as late as 6 months after injury, even in areas with a regenerated endothelium. As a consequence, Ang II–induced DNA replication in injured arteries was greater in the neointima than in the underlying media at all times studied after injury. Although responsiveness to Ang II was suppressed in the media after injury, there was no corresponding change in angiotensin receptor levels in the media at 3 or 10 weeks after injury.
At all times studied after injury, the neointima showed the highest levels in absolute cell growth, ie, in absolute total number of BrdU-labeled cells compared with the normal or injured artery media, in both control and Ang II rats. Because this reflected in large part the greater number of total cells present in the neointima rather than a true evaluation of the growth behavior of the SMCs, the SMC BrdU-labeling fraction (percentage of labeled nuclei) was used to express growth levels. The presence of important differences in baseline labeling fractions between the uninjured and injured arteries at the various times after injury complicated the interpretation of growth data expressed as a "fold increase" over baseline. Thus, the normal carotid artery was used as an internal control for each animal, and the difference in labeling fraction was calculated individually between the injured artery neointima and the media of the contralateral, uninjured carotid artery. Hence, control rats labeled at 3 to 4 weeks after injury showed a higher BrdU-labeling fraction in the neointima than in the contralateral uninjured media, but this difference was increased even further (P<.05) with Ang II infusion. Based on this criterion involving the contralateral normal artery, we conclude that there is an increased responsiveness to Ang II infusion in vivo in the neointima at 3 to 4 weeks but not at later times after injury.
The early increased responsiveness to Ang II in the neointima could represent two nonexclusive mechanisms. First, the overexpression of AT1 receptors could contribute to the higher levels of DNA replication with Ang II, as suggested by their parallel regulation in the neointima over time after injury (Table 1 and Fig 2) and their colocalization at the lumenal surface of the lesion at 3 to 4 weeks22 (Fig 1). There is evidence that Ang II acting via AT1 receptors can increase DNA replication in vascular SMCs independently of BP effects.29 30 31 32 33 Second, the neointima at 3 to 4 weeks exhibits an elevated basal replication rate, and neointimal SMCs are likely to have various cytoplasmic factors required for cell growth. Thus, addition of Ang II to such cells might be expected to have a synergistic effect. Consistent with such a regulation of cellular growth properties, Orlandi et al34 report that the proliferative response to serum is significantly enhanced in SMCs cultured from the neointima of the rat aorta at 2 but not 9 weeks after injury. Other growth-related molecules produced in the neointima may also contribute to the Ang II proliferative effect even in the absence of AT1 receptor overexpression, as in the neointima at 10 weeks (Fig 2). For instance, basic fibroblast growth factor and platelet-derived growth factors AA and BB are expressed at higher levels by the cells at the lumenal surface of the neointima,35 36 37 and these factors potentiate Ang II mitogenic effects29 38 39 or the expression of AT1 receptors40 in cultured SMCs. The causal relationships between SMC replication and AT1 receptor regulation remain incompletely defined.
The present data provide evidence that endothelial regeneration inhibits neointimal growth. Endothelial regeneration correlated with a reduction in the fraction of DNA replicating cells in the neointima in control26 27 as well as Ang II rats. The present data comparing Ang II–induced growth in the neointima with and without endothelium are the first to show the endothelial suppression of an SM growth response in an in vivo model that does not involve an acute response to endothelial denudation. Endothelium-dependent inhibition of DNA replication in SMCs may involve AT1 receptor–mediated release of nitric oxide by ECs.41 In the balloon-injured rat aorta, endothelial regeneration is less extensive at 2 than at 9 weeks following injury, thus raising the possibility that the differences in growth capacities of neointimal cells cultured from this vessel at different times after injury34 may reflect the extent of endothelial denudation of the lesion in vivo. In the present study in the carotid artery, however, we show that the enhanced responsiveness to growth stimulation with Ang II is downregulated with time after injury independently of EC regrowth over the neointima.
An unexpected finding of this study is the loss of responsiveness to systemic Ang II in the media of injured arteries. SMCs in the media showed a diminished replicative response to Ang II by 3 to 4 weeks after injury and no evidence of a mitogenic response to Ang II by 9 to 10 or 27 to 28 weeks. Atrophy of the media is a characteristic histologic feature of arteries with advanced atheroma.42 43 Little is known, however, about the regulation of cell growth in the arterial media underlying intimal thickening. We have reported44 that in spontaneously hypertensive rats arteries with a neointima after balloon injury, as opposed to normal arteries, show no increase in medial thickness with age. Particularly intriguing is the report that SMCs cultured from the arterial media at 2 or 9 weeks after balloon injury show a markedly reduced capacity to proliferate in response to serum stimulation in vitro.34 Although the loss of ability of medial cells to replicate in response to systemic Ang II at 9 to 10 weeks cannot be explained by a corresponding decrease in AT1 receptor concentration, the present data do not imply a general loss of replicative ability in the media. In vitro studies have shown that cultured SMCs are heterogeneous in their ability to undergo DNA synthesis with Ang II, depending, eg, on the balance between proliferative and antiproliferative autocrine factors.32 Thus, during neointimal formation the media may become depleted in cells that replicate DNA in response to Ang II. Alternatively, the possibility that the neointima interfered with Ang II diffusion from the lumen cannot be ruled out. However, the lack of correlation between medial responsiveness and neointimal thickness does not support such a possibility: the injured artery media was responsive to Ang II at 3 to 4 weeks but not later, even though the size of the neointima was comparable at all times. In addition, we have observed45 that Ang II infusion at 5 to 6 weeks after injury does indeed stimulate gene expression (for osteopontin) in the injured artery media, even in the absence of a significant increase in DNA replication in this tissue.
Endogenous catecholamines contribute to the mitogenic effect of Ang II in the normal arterial wall in vivo.24 46 There is no evidence, however, for a direct correlation between the mitogenic effect of Ang II and that of catecholamines in the different SM layers of arteries at various times after injury. First, DNA replication in the neointima at 3 to 4 weeks is not affected by catecholamine infusion46 or by 
1-adrenoreceptor blockade during Ang II infusion.47 Second, 1-agonists stimulate DNA replication in both the media and neointima at 9 to 10 weeks after injury,46 in contrast to the selective neointimal effect seen with Ang II during this later period (Table 1).
Whether Ang II–induced DNA replication resulted in an increase in total SMC number cannot be determined by counting cell nuclei in vessel cross sections, mainly because this method may introduce a systematic sampling bias on the basis of cell nucleus shape, size, and orientation in the arterial wall.48 In another model of Ang II–dependent hypertension, the Goldblatt hypertensive rat shows an increase in SMC polyploidy but not hyperplasia in the thoracic aorta.49 Moreover, SMC number may be regulated by apoptosis (programmed cell death), often in association with increased rates of cell growth, eg, in models of altered blood flow,50 genetic hypertension,51 and vascular wound healing.52 53 Notably, SMC apoptosis is prominent near the lumenal surface of the developing neointima in the rat injured aorta52 and in SMCs found in human coronary atherosclerotic plaques versus normal vessels.53 54 Thus, it is intriguing to speculate that failure to increase neointimal mass with Ang II in the present study may reflect an increase in neointimal SMC apoptosis. Alternatively, the lack of neointimal mass increase may reflect a dose-dependent effect of Ang II since, as we have reported,20 infusion of a higher dose of Ang II (435 ng·kg-1·min-1) at 3 to 4 weeks after injury causes an increase in neointimal mass. Whether Ang II affects SMC apoptosis requires further study.
Consistent with studies testing inhibitors of the renin-angiotensin system against the response to vascular injury,14 15 16 17 18 19 the present study suggests that levels of Ang II are a critical factor during the early phase of neointimal growth after injury, at least in part because of AT1 receptor overexpression in the neointima. Recent evidence that the neointima overexpresses angiotensinogen55 and angiotensin-converting enzyme activity56 57 58 as well as AT1 receptors suggests, but does not establish, a role for endogenous Ang II in the sustained proliferation of neointimal cells in the long term after injury. In summary, we report that SMCs in the different layers of the injured vessel wall show quite distinct patterns of growth response to systemic Ang II in vivo. The selective stimulation of neointimal replication in injured arteries by Ang II further underlines the potential importance of the renin-angiotensin system in the development of vascular lesions.
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Acknowledgments |
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This work was supported in part by a fellowship grant from the Medical Research Council of Canada to Dr deBlois at the Department of Pathology, University of Washington, and by grants from the National Institutes of Health (HL 26405 and HL 42270). We are grateful to Patti Polinski, Ben Rampp, Hillel Schwartz, and Foon Tsui for their excellent technical assistance in surgery and histology and to Marie-France Ross and Andrea Tancredi for their expertise in image analysis.
Received August 11, 1995;
revision received March 20, 1996;
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