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Cardiovascular Research 2000 45(3):777-782; doi:10.1016/S0008-6363(99)00265-5
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Copyright © 2000, European Society of Cardiology

ACE-inhibition promotes apoptosis after balloon injury of rat carotid arteries

Anne Mette Holma,*, Claus Bøgelund Andersenb, Stig Haunsøa and Peter Riis Hansena

aDepartment of Medicine B2142, The Rigshospital, DK-2100 Copenhagen, Denmark
bDepartment of Pathology, The Rigshospital, DK-2100 Copenhagen, Denmark

* Corresponding author. Tel.: +45-35-456-738; fax: +45-35-456-743 amholm{at}hotmail.com

Received 4 June 1999; accepted 27 July 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Angiotensin II stimulates vascular smooth muscle cell (VSMC) growth, and is considered to be an important mediator of intimal thickening after vascular injury. Recent evidence has indicated that VSMC apoptosis plays a major role in the response to balloon injury, and we therefore examined the effect of angiotensin converting enzyme (ACE)-inhibition on VSMC apoptosis and vascular lesion formation in the rat model of balloon injury. Methods: Male Sprague–Dawley rats were subjected to carotid artery balloon injury and randomised to a standard diet or a diet supplemented with 1 mg/ml captopril in the drinking water. Animals were sacrificed 2 and 14 days after injury for assessment of apoptosis and proliferation by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunohistochemistry, respectively. At 14 days post injury, vessel cross-sections were subjected to microscopic morphometry and total cell numbers were determined. Results: At 2 days after balloon injury, captopril-treated animals displayed a significant increase in the percentage of TUNEL-positive VSMCs in the medial area (12±4% vs. 1±1%; P<0.05) as compared to controls. This increase in early apoptosis was associated with decreased intimal cellularity 14 days post injury (238±47 cells/cross-section vs. 449±75 cells/cross-section; P<0.05), and a reduction of neointimal formation (0.13±0.02 mm2 vs. 0.23±0.04 mm2; P<0.05). The fraction of PCNA-positive VSMCs per cross-section 2 or 14 days after injury was not significantly altered by captopril administration. Conclusion: Captopril inhibits neointimal formation in the rat model of arterial injury by mechanisms involving induction of VSMC apoptosis.

KEYWORDS ACE inhibitors; Angiotensin; Apoptosis; Arteries; Restenosis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Treatment with angiotensin-converting enzyme (ACE)-inhibitors [1–4] and angiotensin II (Ang II) receptor antagonists [2,4] has been shown to reduce neointimal formation in the rat model of arterial injury. Consistent with these results, exogenous administration of Ang II can potentiate experimental neointimal formation [5], and balloon injury of the arterial wall may increase the local activity of the renin–angiotensin system e.g., by promoting ACE expression [6] and Ang II receptor subtype I (AT1) binding activity [7]. Ang II has recently been shown to participate in the regulation of apoptosis (programmed cell death) [8–11], which is an important determinant of vessel wall cellularity, neointimal formation and vascular remodelling after balloon injury [12,13]. The present study therefore investigated the effect of ACE-inhibition on vascular lesion formation and apoptosis in the rat model of balloon injury.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Surgery
Male Sprague–Dawley rats (3 months old; average weight 300 g) were randomly divided into two groups that were treated with either 1 mg/ml captopril (Sigma) in the drinking water beginning 6 days before balloon injury or normal drinking water. This drug administration regime has previously been demonstrated to inhibit neointimal formation in the rat model [4]. Captopril was dissolved daily in the drinking water, and the amount of drinking water left in the drinking bottle was measured. The rats had free access to standard rat chow, and could drink water ad libitum. Rats were anaesthetised intraperitoneally with sodium barbital (50 mg/kg), and the endothelium of the left common carotid artery was denuded by three passages of a Fogarty 2F balloon catheter (Baxter Healthcare) as previous described [14,15]. Rats were killed by a sodium barbital overdose 2 or 14 days after injury. The mean blood pressure was measured before treatment and 14 days after surgery under sodium barbital anaesthesia by catherization of the femoral artery with a catheter connected to a pressure transducer (Baxter Healthcare). The investigation conformed with the Guide for the care and use of laboratory animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996).

2.2 Morphometry
The common carotid artery was carefully removed, divided into two segments, fixed in 4% buffered formaldehyde, embedded in paraffin, and 3–5 µm thick cross-sections were cut. Sections were stained for van Gieson Orcein and hematoxylin-eosin. The luminal area (area circumscribed by the intimal border), intimal area (area between the lumen and the internal elastic lamina), medial area (area between the internal and the external elastic lamina), total vessel area (area circumscribed by the external elastic lamina), and intimal to medial (I/M) ratio (intimal area divided by medial area) were determined by computerised digital planimetry, using a videomicroscope with a dedicated image analysing software (Leitz Texture analysing system). To determine the cellularity of the vessel wall, the total number of nuclei in the intimal and medial areas per cross-sections was counted.

2.3 Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL)
Apoptosis was detected in situ on carotid artery cross-sections by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) essentially as previously described [16]. Briefly, cross-sections were dewaxed, rehydrated, and incubated in 20 µg/ml proteinase K (Pharmacia Biotech) for 1 h. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxidase for 5 min. Fragmented DNA were nick end-labelled with a mixture of terminal deoxynucleotidyl transferase (TdT) (22.5 U/section; Pharmacia Biotech), and biotinylated dUTP (0.2 nmol/section; Sigma) in a TdT buffer (30 mM Tris–HCl, 140 mM sodium cacodylate, 1 mM cobalt chloride) for 90 min at 37°C. The reaction was stopped by 15 min incubation in 0.5 M EDTA. The biotinylated fragmented DNA were detected by 25 min incubation in streptavidin conjugated peroxidase followed by 15 min incubation in aminoethyl carbazole. The sections were counterstained with Mayer's hematoxylin and mounted with Kaisers glycerol gelatine. Cells with a brown-red nuclear labelling were defined as TUNEL positive, and a TUNEL index was calculated (100%x[number of TUNEL-positive nuclei/total number of nuclei]). Positive controls were provided by sections pretreated with DNAse I (100 U/ml) (Worthington Biochemical) that demonstrated staining of all nuclei, and sections of mouse small intestine where only the uppermost nuclei at the edges of the crypts facing the lumen were stained [16]. In negative control experiments, TdT was omitted from the labelling mixture, and no staining was detected.

2.4 Transmission electron microscopy
Sections of carotid arteries 2 days after injury were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde and washed in sodium cacodylate buffer. Sections were postfixed in 1% osmium tetroxyde, dehydrated, stained en bloc with 1% uranyl acetate, and embedded in epon. Ultrathin sections were contrasted with lead citrate and the medial area were examined with a Philips 210 electron microscope.

2.5 Proliferating cell nuclear antigen immunohistochemistry
For assessment of proliferation in the vessel wall, cross-sections were stained by immunohistochemistry using an antibody directed against proliferating cell nuclear antigen (PCNA) [17]. In brief, the sections were dewaxed, rehydrated and boiled for 2x5 min in antigen retrieval buffer (Dako) in a microwave, and endogenous peroxidase was blocked by 8 min incubation in 3% hydrogen peroxide. Sections were then incubated with a monoclonal anti-PCNA antibody (Clone PC10, 1:100 dilution; Dako) for 25 min, followed by incubation with a biotinylated rabbit anti-mouse antibody (E464, 1:300 dilution; Dako) for 25 min, and streptavidin conjugated peroxidase (Dako) for 30 min. Aminoethyl carbazole was used as chromogen, sections were counterstained with Mayer's hematoxylin, and mounted with Kaiser glycerol gelatine. Each step was separated by 2x5 min washing in TBS. The number of brown-red labelled PCNA-positive nuclei within each vessel layer was counted, and a PCNA labelling index was calculated [100%x(number of PCNA-positive nuclei/total number of nuclei)].

2.6 Statistical analysis
All data are presented as mean±S.E.M. A two-sided two sample t-test or paired t-test was performed as appropriate. Statistical significance was defined as P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Morphometry
The first part of the protocol included seventeen rats, nine rats in the captopril group and eight rats in the control group, that were subjected to balloon injury, and sacrificed 14 days after injury. The morphological analysis (Fig. 1) demonstrated that captopril treatment significantly reduced neointimal formation (0.13±0.02 mm2 vs. 0.23±0.04 mm2; P<0.05) and I/M ratio (0.96±0.16 vs. 1.71±0.28; P<0.05). In addition, captopril administration decreased blood pressure (99±5 mmHg before treatment vs. 74±4 mmHg 14 days after balloon injury; P<0.05), while no change in blood pressure was observed in the control group (86±4 mmHg before treatment vs. 89±5 mmHg 14 days after balloon injury; NS). Moreover, the daily water consumption was increased in rats treated with captopril (39±2 ml vs. 35±2 ml lost from the drinking bottle each day; P<0.05), corresponding to at most 39±2 mg of captopril/day/animal or a daily oral drug dose of approximately 100 mg/kg.


Figure 1
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  		Fig. 1 Morphometric determination of the total vessel area (area circumscribed by the external elastic lamina), intimal area and medial area of carotid arteries removed 14 days after balloon injury from rats treated with captopril (n=9) or controls (n=8). Columns are means, bars are S.E.M. *P<0.05.

 
3.2 Apoptosis and proliferation
At 2 days post injury i.e., before the appearance of any significant number of neointimal cells, a total of twenty-three rats were sacrificed (ten rats in the captopril group and thirteen rats in the control group). At this early timepoint, the TUNEL index was significantly increased in the media of carotid arteries from rats receiving captopril (12±4% vs. 1±1%; P<0.05) (Figs. 2 and 3Go). Captopril treatment did not affect the TUNEL index 14 days after balloon injury in either the neointima (1.0±0.6% vs. 2.0±0.6%; NS) or the media (0.1±0.5% vs. 0.6±0.5%; NS). TUNEL was not detected in the uninjured carotid arteries in either the control group or the captopril group. Pretreatment of sections with a chelating agent (EDTA) may increase TUNEL specificity in some tissues [18], but use of this step in our TUNEL procedure failed to significantly alter TUNEL parameters (not shown). To provide evidence that the TUNEL-positive VSMCs were apoptotic, carotid sections at 2 days after balloon injury were examined by electron microscopy. In the medial area some VSMCs displayed chromatin condensation that was localised to the edges of the nuclear membrane, cytoplasmic condensation, nuclear shrinking and nucleus fragmentation, all morphological characteristic for VSMC undergoing apoptosis [19] (Fig. 4). Captopril treatment failed to affect the proliferative activity in the injured vessel as indicated by the PCNA labelling index (Fig. 5).


Figure 2
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  		Fig. 2 Proportion of TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labelling] nuclei in carotid arteries sacrificed 2 days (n=10 captopril treated rats, n=13 controls), or 14 days (same rats as in Fig. 1) after balloon injury. Columns are means, bars are S.E.M. *P<0.05.

 

Figure 3
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  		Fig. 3 Representative TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labelling] of carotid arteries 2 days after balloon injury from rats receiving normal drinking water (a–b) or captopril (c–d). (Magnification: a and c, x50, b and d, x150). Note increased number of TUNEL nuclei in the media in rats treated with captopril.

 

Figure 4
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  		Fig. 4 Transmission electron microscopy of carotid arteries 2 days after balloon injury. A viable vascular smooth muscle cell (VSMC) (a), and a apoptotic VSMC (b) in the medial area. Note cytoplasmic condensation and nuclear fragmentation of the apoptotic VSMC (magnification: x3000).

 

Figure 5
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  		Fig. 5 Percentage of PCNA (proliferating cell nuclear antigen)-positive cells in carotid arteries sacrificed 2 days or 14 days after balloon injury (same rats as in Fig. 2). Columns are means, bars are S.E.M.

 
3.3 Lesional cellularity
At 14 days after balloon injury, the reduced neointimal area, in rats receiving captopril, contained a decreased number of cells (238±47 cells/cross-section vs. 449±75 cells/cross-section; P<0.05), while the cellularity was not significantly affected by captopril administration in the media at 2 days (186±28 cells/cross-section vs. 237±22 cells/cross-section; NS) or 14 days after injury (294±19 cells/cross-section vs. 343±30 cells/cross-section; NS).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The present study demonstrates for the first time that inhibition of neointimal formation obtained by ACE-inhibition with captopril in the rat model of arterial injury is associated with increased VSMC apoptosis early but not late after balloon injury. Moreover, administration of captopril reduced neointimal cellularity 14 days post injury, and failed to significantly affect the local proliferative response.

Apoptosis is an active, genetically programmed process by which cells self-destruct, and is characterised by DNA fragmentation into multimers of 180–200 basepairs which may be detected by TUNEL [16]. By essentially representing the (patho)physiological counterpart of cell replication, apoptosis plays a critical role in morphogenesis, tissue homeostasis, and many disease processes [20]. Recently, apoptosis has been documented by TUNEL and electron microscopy in human atherosclerotic and restenotic coronary lesions [12,21], and in the rat model of arterial injury [12,13]. Treatment with different ACE-inhibitors is know to reduce neointimal formation in the rat model of arterial injury [1–4], and as increased cell death is likely to reduce tissue volume, we hypothesised that these agents could favourably influence vascular lesion development by inducing VSMC apoptosis. Our results are in agreement with this contention and they also fall in line with a recent study indicating increased arterial wall VSMC apoptosis in spontaneously hypertensive rats treated with another ACE-inhibitor (enalapril) [10], and the sum of available evidence therefore suggests that the effects we observed with captopril represent a pharmacological class action of ACE-inhibitors [1–4,10]. Ang II binds to 2 major subtypes of receptors, of which the AT1 receptors are thought to induce an anti-apoptotic signal, whereas the AT2 receptors mediate apoptosis [8,11]. AT1 receptors appear to be the dominating Ang II receptor subtype in the arterial media and neointima after balloon injury [7], and in the present study it is therefore likely that, the AT1 receptor subtype was responsible for an anti-apoptotic effect in control rats, which was suppressed by administration of captopril. Although the ACE-inhibitor also reduced systemic blood pressure, this effect is unlikely to have contributed to the reduction of neointimal formation and increased apoptosis. Thus, a previous study has demonstrated that the same degree of blood pressure reduction (induced by verapamil) had no effect on neointimal formation in this model [1], and blood pressure reduction by administration of hydralazine did not affect arterial VSMC apoptosis in spontaneously hypertensive rats [10].

In addition to suppression of Ang II production, the beneficial effect of ACE-inhibition in experimental models of balloon injury appears to be mediated by decreased degradation of kinins, which is associated with increased activity of endogenous nitric oxide (NO) [22,23]. It is well established that administration of NO donors or L-arginine (the physiological precursor of NO) reduces neointimal formation in animal models of arterial injury [24,25], and NO may induce VSMC apoptosis in vitro [26]. Furthermore, Wang et al. recently showed that administration of L-arginine reduced neointimal formation in cholesterol-fed rabbits, and this effect was associated with increased arterial VSMC apoptosis [25]. The possibility that the increase in VSMC apoptosis observed in the present study following treatment with captopril was mediated by reduced degradation of kinins and increased bioactivity of NO therefore deserves further studies.

We found no effect of ACE-inhibition on VSMC proliferation at the selected timepoints (i.e., 2 and 14 days postinjury) as determined by PCNA immunohistochemistry. In this regard, previous studies with ACE-inhibitors in the rat model using thymidine incorporation as a marker of proliferation have yielded discrepant results. Prescott et al. found no effect of benazeprilat on VSMC proliferation 2 days after balloon injury [2], whereas Wong et al. with use of perindopril reported a 25% reduction of VSMC proliferation 2 days after balloon injury, but no effect on the proliferative activity 10 days after injury [3]. Taken together, these results therefore indicate that inhibition of VSMC proliferation probably plays a minor role in the reduction of neointimal formation obtained by ACE-inhibition. In addition, evidence has suggested that the favourable effects of ACE-inhibition on neointimal growth after balloon injury is predominantly caused by reduced VSMC migration and not by inhibition of VSMC proliferation [2]. Increased medial VSMC apoptosis after ACE-inhibition as observed in the current study may explain this effect, since there would be less VSMC available for migration.

It is notable that the TUNEL technique is not exclusively specific for apoptosis, and TUNEL may also occur in necrotic cells and nonapoptotic nuclei with abundant RNA transcription and splicing [27,28]. On the other hand, TUNEL provides an easily accessible histochemical method for the investigation of cell death, which generally appears to be in excellent agreement with other techniques for demonstration of apoptosis in the vessel wall (e.g., electron microscopy or DNA gel electrophoresis) [12,13,21,29]. Furthermore, although PCNA immunohistochemistry compares favourably with thymidine incorporation and bromodeoxyuridine labelling in the rat model [17], the procedure is not completely specific for cell replication and may also label non-diving cells undergoing DNA repair [30].

Unfortunately, attempts to duplicate the favourable effects of ACE-inhibition on vascular lesion formation in the rat model have failed in clinical trials of restenosis prevention after percutaneous transluminal coronary angioplasty (PTCA) [31,32], and this discrepancy is probably explained, in part, by the experimental administration of much higher drug doses [33]. Since the clinical application of a considerably higher systemical dosage of ACE-inhibitors is associated with unacceptable hypotensive side effects, local drug delivery (e.g., by use of intracoronary stents) may represent a future approach for inhibition of vascular lesion formation following PTCA.

In summary, inhibition of neointimal formation by captopril in the rat model of carotid artery balloon injury is associated with increased early VSMC apoptosis and a late reduction of the total number of neointimal VSMC. Induction of apoptosis may represent a new therapeutic strategy for suppression of arterial lesion formation.

Time for primary review 26 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This work was supported by The Danish Heart Foundation, Karen Margrethe Torp-Pedersen and ambassador Emil Torp-Pedersen Foundation, Karen Marie Jørgensen and daughters foundation. AMH is holder of a fellowship from The Danish Medical Research Council. The authors are grateful to Dr. Henning Laursen for his helpful assistance with the morphometric analysis.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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