Cardiovascular Research

Cardiovascular Research 1998 40(1):156-164; doi:10.1016/S0008-6363(98)00127-8
© 1998 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Nelissen-Vrancken, H.J.M. G. Articles by Smits, J. F.M. Search for Related Content PubMed PubMed Citation Articles by Nelissen-Vrancken, H.J.M. G. Articles by Smits, J. F.M. Social Bookmarking          What's this?

Copyright © 1998, European Society of Cardiology

Early captopril treatment inhibits DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts

H.J.Marjorie G. Nelissen-Vrancken^a,*, Marti C. Kuizinga^b, Mat J.A.P. Daemen^b and Jos F.M. Smits^a

^aDepartment of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), Universiteit Maastricht, Maastricht, The Netherlands
^bPathology, Cardiovascular Research Institute Maastricht (CARIM), Universiteit Maastricht, Maastricht, The Netherlands

^* Corresponding author. Tel.: +31 (43) 388 1417; Fax: +31 (43) 367 0940; E-mail: hnel@lmib.azm.nl

Received 8 April 1997; accepted 26 March 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: Cardiac remodeling due to myocardial infarction (MI) includes myocyte hypertrophy, collagen deposition, a rise in DNA synthesis, and normalization of initially diminished maximal coronary bloodflow. Previously, we demonstrated that early captopril treatment can prevent the rise in total DNA synthesis, collagen deposition and hypertrophy. In the present experiments, we investigated the effects of captopril or perindoprilat treatment on cardiac endothelial cell proliferation and maximal coronary flow. Methods: MI was induced by ligation of the left coronary artery in Wistar rats. Sham-operated and infarcted rats were treated with captopril (12 mg/kg.d s.c.) from either day 0–21 (early) or day 21–35 (late) after surgery. In isolated retrogradely perfused rat hearts, maximal coronary flow was determined following maximal dilatation with nitroprusside and adenosine (1 mM each). In separate groups, sections of hearts of sham-operated and MI rats treated with BrdU (day 7–14) and either captopril or perindoprilat (1 mg/kg.d s.c.; day 0–14) were double stained with a monoclonal anti-BrdU antibody and the lectin GSI. The total fraction of DNA synthesizing cells and its proportion of endothelial cells was determined. Results: Maximal coronary flow was completely normalized in MI hearts within three weeks after surgery. Early captopril, but not late captopril, inhibited the normalization of maximal coronary flow in MI hearts (Early: sham, 27.4±1.0; MI, 21.2±1.4 ml/min; P<0.05; mean±SEM) without affecting the hypertrophic response. The total fraction of DNA synthesizing cells was significantly increased in MI hearts (sham: 7.6±1.9; MI: 14.9±2.2%). The proportion of endothelial cells, however, was comparable in sham-operated and infarcted hearts (sham: 30±3; MI: 33±3%). Both early captopril and perindoprilat treatment inhibited total DNA synthesis in MI hearts. Only in captopril pre-treated hearts, this inhibition was associated with a disproportionate inhibition of the endothelial cell proliferation (10.3±2.0%). Conclusion: Early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the renin angiotensin system.

KEYWORDS Rat; Myocardial infarction; Captopril; Perindoprilat; Endothelial cells; Coronary blood vessels

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Induction of myocardial infarction (MI) induces remodeling of both the infarcted and the non-infarcted regions of the heart [1]. Structural remodeling in the infarcted part results predominantly in dilatation, scarring, and thinning of the infarcted tissue, whereas remodeling of the non-infarcted part of the ventricles includes hypertrophy of the myocytes, and an increase in collagen content [1–4]. These changes in the non-infarcted part are associated with an early increase in DNA synthesis [3], which is predominantly localized in fibroblasts and endothelial cells [5]. Furthermore, total maximal coronary flow normalizes within 5 weeks after MI due to a combination of recruitment of pre-existing coronary vessels and growth of new vessels [6].

The cardiac renin–angiotensin system (RAS) plays an important role in the remodeling processes of the heart. With respect to angiotensin converting enzyme (ACE), both increased activity and mRNA expression were observed in rat hearts with pressure overload-induced hypertrophy [7], in the right ventricle and the septum of infarcted rat hearts [8], and in the left ventricle of failing human hearts [9]. Similarly, we previously observed an increase in mRNA expression in the infarcted rat heart [10].

Late captopril treatment (week 3–5 following MI) significantly improved cardiac function [11, 12], and increased the 1 year survival rate of MI rats [13]. It had no effect on cardiac collagen deposition and DNA synthesis [3]. In contrast, early captopril treatment (week 0–3 following MI) worsened cardiac function in MI rats [11]. This negative effect on cardiac function was associated with inhibition of the rise in total DNA synthesis and collagen deposition [3]. It is, however, not known whether captopril treatment also affects the previously observed normalization of maximal coronary flow, which is due to a combination of recruitment of pre-existing coronary vessels and growth of new vessels [6]. To investigate the effect of captopril treatment on coronary vessel growth in infarcted hearts, both endothelial cell proliferation, as an index for vessel growth, and maximal coronary flow were quantified in sham-operated and infarcted hearts following early captopril treatment. Endothelial cell proliferation was measured at day 14, because DNA synthesis is maximal between day 7 and 14 and returns to baseline within 3 weeks [3]. Because the outcome of the proliferative response (i.e. vessel growth) should be expected to be complete at 3 weeks after MI, which agrees with our observations [6], the effect of captopril treatment on this normalization was investigated at day 21. In addition, maximal coronary flow was investigated in sham-operated and infarcted hearts following late captopril treatment (day 21–35). To evaluate the ACE specificity of the captopril effects, also total proliferation and endothelial cell proliferation were investigated following early perindoprilat treatment.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animals
Male Wistar rats (Harlan-Winkelmann, Borchen, Germany), weighing 280–320 g, were used. Animals had free access to standard food (Hope Farms, Woerden, the Netherlands) and tap water, and were housed in groups of 2–4 rats. The experimental procedures were approved by the Ethical Committee for the Use of Experimental Animals of the institution, and conform with the Guide for the Care and the Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).

2.2 Myocardial infarction surgery
Myocardial infarction (MI) was induced by coronary artery ligation under pentobarbital anesthesia (60 mg/kg i.p.) [14, 15]. Intraoperatively, the animals were respirated with room air (60 strokes/min, tidal volume 3 ml) after the trachea was intubated. After thoracotomy in the fourth left intercostal space, the heart was exteriorized and a 6-0 silk suture was passed under the left anterior descending coronary artery (LAD) near the origin of the pulmonary artery. In sham-operated animals the suture was looped through the myocardium next to the LAD. After the heart was returned to its normal position, the suture was tied. The ribs were pulled together with 3-0 silk, and the skin was sutured. Animals were allowed to recover for 14 days in histological experiments and for 21 or 35 days in perfusion experiments.

2.3 Perfusion experiments
2.3.1 Treatment
Osmotic mimipumps were implanted subcutaneously in the neck under ether anesthesia and rats were infused with captopril (12 mg/kg.d s.c., Alzet 2001; Alza, Palo Alto, USA). Early captopril treatment (day 0–21) started immediately after sham or MI surgery and was continued for 3 weeks. Late captopril treatment (day 21–35) started 3 weeks after sham or MI surgery and captopril was infused for 2 weeks. Osmotic minipumps were replaced every week under ether anesthesia.

2.3.2 Isolated heart perfusion
Isolated hearts were perfused as described previously [6, 16]. Under pentobarbital anesthesia (60 mg/kg i.p.), hearts of MI and sham-operated animals (day 21 or 35) were rapidly excised and immediately immersed in ice-chilled perfusion medium (see below). After removal of lung and fat tissue, hearts were connected to the aortic cannula of the perfusion system and retrograde perfusion (Langendorff perfusion model) was started at a perfusion pressure of 60 mmHg.

The hearts were perfused with a modified Krebs-Henseleit solution (mM: NaCl, 130; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 1.2; NaH₂PO₄, 1.2; NaHCO₃, 25.0; glucose, 10.0; pyruvate, 5.0). The solution was maintained at 37°C, gassed with 95% O₂ and 5% CO₂ to obtain a pH of 7.4, and continuously filtered (1.2 µm Millipore filter) throughout the perfusion period. The hearts were paced at 5 Hz.

Coronary flow was measured by an electromagnetic flow probe (Skalar, Delft, the Netherlands) mounted in the aortic inflow tract. Perfusion pressure was measured by a pressure transducer (Gould Spectramed DTX+, Spectramed) connected to the inflow of the aortic canula. Both variables were continuously monitored on-line by a computer (486 DX2; 40 MHz) using a hemodynamic data acquisition system (HDAS; Instrumental Services, Universiteit Maastricht, the Netherlands).

2.3.3 Perfusion measurements
The hearts were prepared for retrograde perfusion at day 21 (control and early captopril) or day 35 (control and late captopril) after sham surgery or induction of MI. After equilibration of the isolated hearts, basal values of coronary flow were determined. Thereafter, coronary vasodilatation was induced by subsequent injections of 0.5 ml of adenosine (1 mM), nitroprusside (1 mM) and adenosine+nitroprusside (1 mM each). Maximal coronary flow was defined as smallest resistance observed.

2.3.4 Measurement of infarct size
To measure infarct size, the hearts were cut into transverse slices of 1–2 mm, resulting in 4 slices. In previous experiments, we investigated the correlation between the mean infarct size and the infarct size at four different levels (level 1 is basal level; level 4 is apical level). As demonstrated in Fig. 1, there was a very good correlation between the mean infarct size and the infarct size at level 2 (mid-ventricular slice). Therefore, the mid-ventricular slice was fixed with formalin and embedded in paraffin, whereafter transverse sections (4 µm) were stained according to the modified AZAN technique [3]. Infarct size was determined by computerized morphometry (Quantimet 570, Leica, Cambridge, UK) and expressed in percentage of left ventricular circumference [10].

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Correlation between mean infarct size (calculated as the mean of the infarct sizes at all levels) and infarct size at the individual levels of the heart. Level 1 is the basal level, and level 4 is the apical level. The straight line represents the correlation between infarct size at level and mean infarct size. The dotted lines represent the 95% confidence intervals. There is a very good correlation between infarct size at level 2 (mid-ventricular slice) and mean infarct size (r2=0.960).

 
2.4 Structural experiments
2.4.1 Captopril treatment
Osmotic mimipumps were implanted subcutaneously in the neck under ether anesthesia and sham-operated and MI rats were infused with captopril (12 mg/kg.d s.c., Alzet 2001) from day 0 to 14. Osmotic minipumps were replaced after one week under ether anesthesia.

2.4.2 Perindoprilat treatment
Osmotic mimipumps were implanted subcutaneously in the neck under ether anesthesia and sham-operated and MI rats were infused with perindoprilat (1 mg/kg.d s.c., Alzet 2002; a generous gift from dr. E. Scalbert, Servier, Paris, France) from day 0 to 14. In pilot experiments, the doses used of perindoprilat and captopril resulted in a comparable shift of the angiotensin I dose–pressure curve (data not shown).

2.4.3 BrdU infusion
Osmotic minipumps were implanted subcutaneously in the neck under ether anesthesia, and sham-operated and infarcted rats were infused with BrdU (0.8 mg/kg.d s.c., Alzet 2001) from day 7 to 14.

2.4.4 Double staining of heart sections
At day 14, the rats were anesthetized with ether. The hearts of BrdU treated rats were arrested in diastole by injecting 2 ml 0.1 M CdCl₂ into the inferior caval vein and perfused via a catheter in the abdominal aorta with phosphate buffered saline (PBS, pH 7.4), containing 0.5 mg/ml nitroprusside (Hoffmann-La Roche, Mijdrecht, the Netherlands) for 5–10 min at a pressure of approximately 100 mmHg. Thereafter, the hearts were perfusion-fixed for 10 min with 10% phosphate buffered formalin (1:1 diluted in PBS), containing 0.5 mg/ml nitroprusside. The hearts were removed and stored in 10% phosphate buffered formalin for 24 h at room temperature. Subsequently, the hearts were cut into transverse slices of 4 mm, processed and embedded in paraffin.

To discriminate between DNA synthesizing endothelial cells and non-endothelial cells in the non-infarcted part of the myocardium, transverse sections (4 µm) were double stained with the lectin Griffonia Simplicifolia I (GSI; Sigma, St. Louis, USA) and a monoclonal anti-BrdU antibody (Eurodiagnostics, Apeldoorn, the Netherlands), as previously described [5]. Therefore, the sections were dewaxed and rehydrated, and endogenous peroxidase was inhibited by methanol/H₂O₂ (0.3%) for 15 min. The sections were incubated overnight with the biotinylated lectin GSI (1:100) at room temperature, followed by incubation with the alkaline phosphatase conjugated biotin–avidin complex (1:200; Dakopatts, Glostrup, Denmark) for 30 min at room temperature and development with Fast Blue BB' (Sigma, St. Louis, USA). The staining of BrdU incorporation with a murine monoclonal anti-BrdU antibody was performed as described previously [3]. A representative picture of the double staining is presented in Fig. 2.

View larger version (171K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Representative picture of a heart section double stained with the lectin Griffonia Simplicifolia I (GSI) and the monoclonal anti-BrdU antibody. Brown coloured dots: BrdU positive nuclei; blue coloured lines: endothelial cells; arrow: BrdU positive endothelial cell, representing a DNA synthesizing endothelial cell. Magnification: 400x.

 
To quantify the total number of DNA synthesizing cells, parallel sections were stained with the monoclonal anti-BrdU antibody only (single staining).

2.4.5 Measurements
Total DNA synthesizing cells and the amount of DNA synthesizing endothelial cells were determined in hearts of untreated and treated sham-operated and infarcted rats. The origin of all samples was blinded to the investigators. Cell numbers were determined microscopically with an eyepiece grid (400x magnification), as described previously [3]. All measurements were performed by two experienced investigators. Intra- and inter-observer variations were less than 10%.

DNA synthesizing endothelial cells in the subendocardial region of the left ventricle (septum and papillary muscle) were identified based on the co-localisation of both BrdU positive nuclei and GSI stained microvascular profiles. For the single BrdU staining a total of 700–1300 cells per heart were counted, whereas for the double staining at least 500 BrdU positive nuclei were counted.

The percentage of BrdU positive nuclei was calculated from the number of BrdU positive cells and the total number of cells. The percentage of BrdU positive endothelial cells was calculated from the number of BrdU positive endothelial cells and the total number of BrdU positive cells.

Infarct size was determined in the mid-ventricular slice, as described above.

2.5 Data analysis
Only hearts with infarct sizes >21% were used in the MI groups, since smaller infarcts do not have detectable hemodynamic consequences in vivo [11].

Data from all experiments were compared by one-way analysis of variance followed by a post-hoc test. For comparison of groups to untreated sham animals, we used Dunnett's test, defining untreated sham rats as controls. For all other comparisons, we employed a Bonferroni procedure [17]. Data were expressed as mean±SEM. Differences were regarded to be statistically significant at a value of P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Perfusion experiments
Induction of MI resulted in an increase in heart weight, although only significantly at day 35 (Table 1). Neither early nor late captopril treatment affected body and heart weight. Infarct sizes were comparable in the groups.

View this table: [in this window] [in a new window]   Table 1 Body weight, heart weight and infarct size of untreated and treated sham-operated (sham) and infarcted (MI) rats in perfusion experiments

 
Basal coronary flow was comparable in sham-operated and infarcted hearts at days 21 and 35, and was not affected by captopril treatment (data not shown). Although more than 40% of the left ventricle was infarcted (Table 1), there were no significant differences between maximal coronary flow in untreated sham-operated and infarcted hearts at days 21 and 35 following surgery (Fig. 3). Both in sham-operated and infarcted hearts, maximal coronary flow was unaffected by late captopril treatment (day 21–35). In contrast, early captopril treatment (day 0–21) inhibited the normalization of maximal coronary flow in infarcted hearts. In sham-operated rats, captopril treatment had no significant effect on maximal coronary flow.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Maximal coronary flow of untreated (open bars) and captopril treated (cross-hatched bars) sham-operated (sham) hearts, and of untreated (filled bars) and captopril treated (fine cross-hatched bars) infarcted (MI) hearts at day 21 and 35 after surgery. (*P<0.05 compared to captopril treated sham-operated hearts). Data are expressed as mean±SEM.

 
3.2 Structural experiments
The total labeling fraction of DNA synthesizing cells in sham-operated hearts was 7.6±1.9% (Fig. 4A). Of these cells 30±3% were identified as endothelial cells (Fig. 4B). Induction of MI almost doubled the total DNA synthesizing cell fraction at day 14 after surgery (14.9±2.2%; Fig. 4A). The proportion of endothelial cells of this population was comparable to that in sham-operated hearts (33±3%; Fig. 4B). Thus, a proportional increase in the number of BrdU-positive endothelial cells and non-endothelial interstitial cells accounts for the increase in total amount of DNA synthesizing cells.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Total BrdU labeling fraction (A) and the percentage of labeled endothelial cells of the total labeling fraction (B) in untreated sham-operated (sham; open bars) and infarcted (MI; filled bars) hearts, in captopril treated sham-operated (cross-hatched bars) and MI hearts (fine cross-hatched bars), and in perindoprilat treated sham-operated (hatched bars) and MI hearts (fine hatched bars). (*P<0.05 compared to untreated sham-operated hearts; +P<0.05 compared to untreated MI hearts). Data are expressed as mean±SEM.

 
Early captopril treatment (day 0–14) inhibited total DNA synthesis both in sham-operated (4.0±0.5%) and MI hearts (7.8±1.5%; Fig. 4A). As shown in Fig. 4B, the inhibition of DNA synthesis by early captopril treatment had no effect on the proportion of endothelial cells that DNA synthesized in sham-operated hearts (32.0±1.2%), but was associated with a disproportionate inhibition of DNA synthesis in endothelial cells in MI hearts (10.3±2.0%).

Early perindoprilat treatment (day 0–14) inhibited total DNA synthesis in MI hearts (Fig. 4A). In contrast to captopril, the inhibition of DNA synthesis by early perindoprilat tretament had no effect on the proportion of endothelial cells that DNA synthesized in MI hearts (32.7±1.3%; Fig. 4B).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In previous experiments, we demonstrated that following MI remodeling processes in the non-infarcted part of the heart are characterized by an increase in collagen content, and an early increase in DNA synthesis, which is mainly confined to fibroblasts and endothelial cells (reviewed in ref. [1]). Recently, we observed a complete normalization of maximal coronary flow within 5 weeks after MI due to a combination of recruitment of pre-existing vessels and growth of new coronary vessels in the borderzone between infarcted and surviving myocardium [6]. The increase in collagen content and the early rise in (total) DNA synthesis following MI could be prevented by early (0–3 weeks) captopril treatment [3]. In the present experiments, we investigated the effect of chronic captopril treatment on endothelial cell DNA synthesis and its possible functional consequences on maximal coronary flow.

Maximal coronary flow was comparable in hearts of untreated sham-operated and infarcted rats both at days 21 and 35, which confirms our previous findings [6]. Late captopril treatment did not affect maximal coronary flow. In contrast, early captopril treatment significantly decreased maximal coronary flow in infarcted hearts but not in sham-operated hearts. Moreover, total DNA synthesis was inhibited in sham-operated and MI hearts following early captopril treatment, which was associated with a disproportionate inhibition of endothelial cell proliferation. The inhibition of total DNA synthesis in MI hearts following early perindoprilat treatment, however, was associated with a proportional inhibition of endothelial cell proliferation. These experiments clearly indicate that early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the RAS.

In the present experiments, the hypertrophic response following induction of MI was not inhibited by captopril. This is in contrast with the general believe that captopril prevents cardiac hypertrophy. In literature, however, the observations concerning a regression of cardiac hypertrophy following captopril treatment in infarcted rats are not consistent. Several studies demonstrated no regression of cardiac hypertrophy following late captopril treatment [11, 18, 19], whereas others showed a diminished heart weight in rats with large infarct sizes [12, 20]. Also following early captopril treatment, the observations are not consistent. Both a regression [3, 11, 21, 22]and no regression of cardiac weight [23–26]have been observed. The reason for this inconsistency is not clear, but does not seem to depend on doses used and/or duration of therapy. Due to unaltered heart weights in captopril pre-treated rats, the observed decrease in maximal coronary is not caused by a diminshed heart weight in the present experiment.

Metsärinne and colleagues [27]demonstrated an inhibitory effect of angiotensin II on rat coronary endothelial cell proliferation in vitro. In the presence of the AT₂-receptor antagonist PD123177, however, angiotensin II induced proliferation of these cells, whereas the addition of the AT₁-receptor antagonist losartan reversed this effect [28]. In contrast to these observations in cell culture, we recently observed a partial inhibition of DNA synthesis in infarcted rat hearts following early treatment with the AT₂-antagonist PD123319 [29].

Thus far, the influence of the RAS on coronary vascularization and coronary flow has predominantly been investigated in hypertensive patients and hypertensive animal models. In hypertension, the increase in vascularization is mainly secondary to hypertrophic growth of the myocytes and to a lesser extent to normal tissue growth. The increase in vascular density or coronary flow, however, is not proportional to the hypertrophic growth response [30–33], although the increase in coronary flow seems to depend on the duration of hypertension [34, 35]. Long-term treatment with the ACE inhibitor enalapril decreased minimal coronary resistance and increased maximal coronary flow and flow reserve in hypertensive patients with angina pectoris [36]. In hypertension induced by aortic-banding, 4-weeks of captopril treatment decreased minimal coronary resistance, and thus increased maximal coronary flow [23]. Long-term ramipril treatment (20 week; treatment started in utero) of spontaneously hypertensive rats (SHR) increased myocardial capillary length density, independent of blood pressure reduction or inhibition of development of hypertrophy [37]. In the cremaster muscle, however, an inhibition of vascular growth has been observed following long-term captopril treatment in one-kidney, one clip hypertensive rats [38]. Thus, in hypertensive patients and in most hypertensive animals, long-term ACE inhibition enhances maximal coronary flow, due to a more pronounced outgrowth of the coronary vascularization. The observations in hypertension are in contrast to observations in non-hypertensive models. Angiogenic responses to angiotensin II have been described in chick chorioallantoic membrane [39], in dog kidney [40], and rabbit cornea [41]. Furthermore, Munzenmaier and Greene [42]observed an increase in vessel density in rat cremaster muscle following a subpressor dose of angiotensin II. In a rat model for chronic peripheral ischemia, we previously observed an abolishment of the increase in capillarization in soleus muscles by ACE inhibitors [43]. The observations in this latter model of ischemia are consistent with the observations in the present chronic ischemia model. The discrepancy between hypertensive models and severely ischemic models suggests that the role of the RAS in vessel growth may depend on the pathophysiological condition.

The inhibition of endothelial cell proliferation and coronary vessel growth following early ACE inhibition may depend on the blood pressure lowering effect of the ACE inhibitor. Late captopril treatment, however, did not prevent the normalization in maximal coronary flow, despite a comparable decrease in blood pressure [11]. Furthermore, chronic treatment with the vasodilator hydralazine did not lower total DNA synthesis in MI hearts [3], suggesting a lack of involvement of blood pressure reduction in the present observations. The contribution of elevated bradykinin levels due to inhibition of its degradation can not be ruled out. Thus far, however, only a stimulatory rather than an inhibitory effect of bradykinin on endothelial cell proliferation in vitro [44]and on myocardial capillary growth in hypertensive rats [45]has been described.

A more likely explanation for the present observations is the prevention of (local) angiotensin II formation by inhibition of ACE. Munzenmaier and Greene observed an increase in vessel density in rat cremaster muscle following a subpressor dose of angiotensin II, which was enhanced following co-infusion of the AT₂-antagonist PD123319 and inhibited following co-infusion of the AT₁-antagonist losartan [42]. In cultured rat coronary endothelial cells, angiotensin II inhibits growth [27, 28]. In accordance with the observations of Munzenmaier and Greene [42], this inhibitory response depended upon AT₂-receptor stimulation, since addition of the AT₂-antagonist PD123177 resulted in a stimulation of proliferation. Thus, in vivo the balance may be in favour of (AT₁-mediated) proliferation, whereas in vitro, inhibition (AT₂-mediated) may be favoured. In infarcted hearts, we recently observed a partial inhibition of DNA synthesis following early treatment with the AT₂-antagonist PD123319 [29].

In contrast to early treatment with perindoprilat or with the AT₂-antagonist PD123319 [29], captopril treatment resulted in a disproportionate inhibition of proliferating endothelial cells in hearts of infarcted rats. This suggests that in addition to the effect on the RAS, captopril may influence other angiogenic factors involved in coronary vascular remodeling following MI, like VEGF, FGF, TGF-, IGF-I, and PDGF [46].

Previously, we observed a worsening of cardiac function in infarcted rats following early captopril treatment [11]. The inhibition of e.g. cardiac collagen deposition in the early phase following MI was assumed to be responsible for the deleterious effect of early captopril treatment [1]. The present observations suggest that also the prevention of endothelial DNA synthesis and normalization of maximal coronary flow contributes to the reduction of cardiac function in infarcted rats following early captopril treatment.

The present study employed functional and structural data to obtain an indication of endothelial cell proliferation following MI, its effect on the coronary system, and the effect of ACE inhibition thereupon. For technical reasons, we chose the septum and papillary muscles for the quantitation of BrdU/lectin staining. In contrast, maximal coronary flow is obviously an overall measure. There may be regionally different adaptive mechanisms in the healthy ventricle versus the borderzone. Our functional methods do not allow to differentiate between the effects on coronary vessels in septum, borderzone and infarcted area. However, we previously demonstrated a very low coronary flow in the infarcted area [6]. Therefore, it is not likely that blood vessels in the infarct zone contribute to the observed effects in a significant manner. On the other hand, the borderzone may be an interesting area, because coronary blood flow following MI was enhanced in this area [6]. We tried to quantify total DNA synthesis and endothelial cell proliferation in the borderzone of MI hearts, but no reproducible data could be obtained. Nonetheless, we demonstrated both a diminished endothelial cell proliferation in the healthy part of the MI heart and a diminished maximal coronary flow. This suggests that the alterations observed in the healthy part of the infarcted heart substantially contribute to the maximal coronary blood flow.

In conclusion, early captopril treatment inhibits endothelial cell proliferation and normalization of maximal coronary flow, which suggests inhibition of coronary vessel growth in infarcted rat hearts. The present experiments suggest that the RAS plays a prominent role in the coronary vessel growth as part of the remodeling processes in infarcted hearts. Considering that these processes are beneficial, inhibition of cardiac ACE in the early phase after myocardial infarction may inhibit the functional adaptation of the infarcted heart. Besides inhibition of cardiac ACE, captopril may also influence other angiogenic factors involved in the coronary vascular remodeling following MI.

Time for primary review 29 days

	Acknowledgements

 
This work was supported by grant 902-18-291 from the Dutch Heart Foundation and NWO (the Netherlands). We thank Elsbeth A. Raes and Anique J.M.H. Janssen for expert technical assistance.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Smits J.F.M., Passier R.C.J.J., Nelissen-Vrancken H.J.M.G., Cleutjens J.P.M., Kuizinga M.C., Daemen M.J.A.P. Does ACE inhibition limit structural changes in the heart following myocardial infarction? Eur Heart J (1995) 16(Suppl. N):46–51.[CrossRef][Web of Science][Medline]
2. Anversa P., Beghi C., Kikkawa Y., Olivetti G. Myocardial infarction in rats. Infarct size, myocyte hypertrophy, and capillary growth. Circ Res (1986) 58:26–37.[Abstract/Free Full Text]
3. van Krimpen C., Smits J.F.M., Cleutjens J.P.M., et al. DNA synthesis in the non-infarcted cardiac interstitium after left coronary artery ligation in the rat: effects of captopril. J Mol Cell Cardiol (1991) 23:1245–1253.[CrossRef][Web of Science][Medline]
4. Cleutjens J.P.M., Verluyten M.J.A., Smits J.F.M., Daemen M.J.A.P. Collagen remodeling after myocardial infarction in the rat heart. Am J Pathol (1995) 147:325–338.[Abstract]
5. Kuizinga M.C., Cleutjens J.P.M., Smits J.F.M., Daemen M.J.A.P. Griffonia simplicifolia I (GSI): a suitable rat cardiac microvascular marker on paraffin embedded tissue. J Mol Cell Cardiol (1992) 24(Suppl. V):S57.
6. Nelissen-Vrancken H.J.M.G., Debets J.J.M., Schnoeckx L.H.E.H., Daemen M.J.A.P., Smits J.F.M. Time-related normalization of maximal coronary flow in isolated perfused hearts of rats with myocardial infarction. Circulation (1996) 93:349–355.[Abstract/Free Full Text]
7. Schunkert H., Dzau V.J., Tang S.S., Hirsch A.T., Apstein C.S., Lorell B.H. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation. J Clin Invest (1990) 86:1913–1920.[Web of Science][Medline]
8. Hirsch A.T., Talsness C.E., Schunkert H., Paul M., Dzau V.J. Tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure. Circ Res (1991) 69:475–482.[Abstract/Free Full Text]
9. Studer R., Reinecke H., Muller B., Holtz J., Just H., Drexler H. Increased angiotensin-I converting enzyme gene expression in the failing human heart. Quantification by competitive RNA polymerase chain reaction. J Clin Invest (1994) 94:301–310.[Web of Science][Medline]
10. Passier R.C.J.J., Smits J.F.M., Verluyten M.J.A., Studer R., Drexler H., Daemen M.J.A.P. Activation of angiotensin-converting-enzyme expression in the infarct zone following myocardial infarction. Am J Physiol (1995) 269:H1268–H1276.[Web of Science][Medline]
11. Schoemaker R.G., Debets J.J.M., Struyker-Boudier H.A.J., Smits J.F.M. Delayed but not immediate captopril therapy improves cardiac function in conscious rats, following myocardial infarction. J Mol Cell Cardiol (1991) 23:187–197.[CrossRef][Web of Science][Medline]
12. Litwin S.E., Litwin C.M., Raya T.E., Warner A.L., Goldman S. Contractility and stiffness of noninfarcted myocardium after coronary ligation in rats. Effects of chronic angiotensin converting enzyme inhibition. Circulation (1991) 83:1028–1037.[Abstract/Free Full Text]
13. Pfeffer M.A., Pfeffer J.M., Steinberg C., Finn P. Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation (1985) 72:406–412.[Abstract/Free Full Text]
14. Pfeffer M.A., Pfeffer J.M., Fishbein M.C., et al. Myocardial infarct size and ventricular function in rats. Circ Res (1979) 44:503–512.[Abstract/Free Full Text]
15. Schoemaker R.G., Debets J.J.M., Struyker-Boudier H.A.J., Smits J.F.M. Beneficial hemodynamic effects of two weeks' milrinone treatment in conscious rats with heart failure. Eur J Pharmacol (1990) 182:527–535.[CrossRef][Web of Science][Medline]
16. Snoeckx L.H.E.H., Van der Vusse G.J., Coumans W.A., Willemsen P.H.M., Van der Nagel T., Reneman R.S. Myocardial function in normal and spontaneously hypertensive rats during reperfusion after a period of global ischaemia. Cardiovasc Res (1986) 20:67–75.[Abstract/Free Full Text]
17. Wallenstein S., Zucker C.O.L., Fleiss J.L. Some statistical methods useful in circulation research. Circ Res (1980) 47:1–9.[Abstract/Free Full Text]
18. Raya T.E., Fonken S.J., Lee R.W., et al. Hemodynamic effects of direct angiotensin II blockade compared to converting enzyme inhibition in rat model of heart failure. Am J Hypertens (1991) 4:334S–340S.[Medline]
19. Hirsch A.T., Talsness C.E., Smith A.D., Schunkert H., Ingelfinger J.R., Dzau V.J. Differential effects of captopril and enalapril on tissue renin–angiotensin systems in experimental heart failure. Circulation (1992) 86:1566–1574.[Abstract/Free Full Text]
20. Litwin S.E., Raya T.E., Warner A., Litwin C.M., Goldman S. Effects of captopril on contractility after myocardial infarction: experimental observations. Am J Cardiol (1991) 68:26D–34D.[Medline]
21. Bralet J., Marie C., Mossiat C., Lecomte J.-M., Gros C., Schwartz J.-C. Effects of alatriopril, a mixed inhibitor of atriopeptidase and angiotensin I converting enzyme, on cardiac hypertrophy and hormonal responses in rats with myocardial infarction. Comparison with captopril. J Pharmacol Exp Ther (1994) 270:8–14.[Abstract/Free Full Text]
22. Bélichard P., Savard P., Cardinal R., et al. Markedly different effects on ventricular remodeling result in a decrease in inducibility of ventricular arrhythmias. J Am Coll Cardiol (1994) 23:505–513.[Abstract]
23. Regan C.P., Anderson P.G., Bishop S.P., Berecek K.H. Captopril prevents vascular and fibrotic changes but not cardiac hypertrophy in aortic-banded rats. Am J Physiol (1996) 271:H906–H913.[Medline]
24. Capasso J.M., Anversa P. Mechanical performance of spared myocytes after myocardial infarction in rats: effects of captopril treatment. Am J Physiol (1992) 263:H841–H849.[Web of Science][Medline]
25. Mill J.G., Gomes A.P.V., Carrara A.B., Gomes M.G.S., Vassallo D.V. Influence of chronic captopril therapy on the mechanical performance of the infarcted rat heart. Pharmacol Res (1994) 29:77–88.[CrossRef][Web of Science][Medline]
26. Capasso J.M., Li P., Meggs L.G., Herman M.V., Anversa P. Efficacy of angiotensin-converting enzyme inhibition and AT1 receptor blockade on cardiac pump performance after myocardial infarction in rats. J Cardiovasc Pharmacol (1994) 23:584–593.[Web of Science][Medline]
27. Metsärinne K.P., Stoll M., Gohlke P., Paul M., Unger T. Angiotensin II is antiproliferative for coronary endothelial cells in vitro. Pharm Pharmacol Lett (1992) 2:150–152.
28. Stoll M., Steckelings M., Paul M., Bottari S.P., Metzger R., Unger T. The angiotensin AT₂-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest (1995) 95:651–657.[Web of Science][Medline]
29. Kuizinga MC, Smits JFM, Arends JW, Daemen MJAP. AT2 receptor blockade reduces cardiac interstitial cell DNA synthesis and cardiac function after rat myocardial infarction. J Mol Cell Cardiol 1998; in press.
30. Vitullo J.C., Penn M.S., Rakunsan K., Wicker P. Effects of hypertension and aging on coronary arteriolar density. Hypertension (1993) 21:406–414.[Abstract/Free Full Text]
31. Tomanek R.J. Capillary and pre-capillary coronary vascular growth during left ventricular hypertrophy. Can J Physiol Pharmacol (1986) 2:114–119.
32. Anversa P., Ricci R., Olivetti G. Coronary capillaries during normal and pathological growth. Can J Physiol Pharmacol (1986) 2:104–113.
33. Mall G., Zimmer G., Baden S., Mattfeldt T. Capillary neoformation in the rat heart-stereological studies on papillary muscles in hypertrophy and physiologic growth. Basic Res Cardiol (1990) 85:531–540.[CrossRef][Web of Science][Medline]
34. Tomanek R.J., Schalk K.A., Marcus M.L., Harrison D.G. Coronary angiogenesis during long-term hypertension and left ventricular hypertrophy in dogs. Circ Res (1989) 65:352–359.[Abstract/Free Full Text]
35. O'Keefe D.D., Hoffman J.I.E., Cheitlin R., O'Neill M.J., Allard J.R., Shapkin E. Coronary blood flow in experimental canine left ventricle hypertrophy. Circ Res (1978) 43:43–51.[Abstract/Free Full Text]
36. Motz W., Strauer B.E. Improvement of coronary flow reserve after long-term therapy with enalapril. Hypertension (1996) 27:1031–1038.[Abstract/Free Full Text]
37. Unger T., Mattfeldt T., Lamberty V., et al. Effect of early onset angiotensin converting enzyme inhibition on myocardial capillaries. Hypertension (1992) 20:478–482.[Abstract/Free Full Text]
38. Wang D.-H., Prewitt R.L. Captopril reduces aortic and microvascular growth in hypertensive and normotensive rats. Hypertension (1990) 15:68–77.[Abstract/Free Full Text]
39. Le Noble F.A.C., Hekking J.W.M., Van Straaten H.W.M., Slaaf D.W., Struyker-Boudier H.A.J. Angiotensin II stimulates angiogenesis in the chorio-allantoic membrane of the chick embryo. Eur J Pharmacol (1991) 195:305–306.[CrossRef][Web of Science][Medline]
40. Fernandez L.A., Caride V.J., Twickler J., Galardy R.E. Renin–angiotensin and development of collateral circulation after renal ischemia. Am J Physiol (1982) 243:H869–H875.[Web of Science][Medline]
41. Fernandez L.A., Twickler J., Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med (1985) 105:141–145.[Web of Science][Medline]
42. Munzenmaier D.H., Greene A.S. Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension (1996) 27:760–765.[Abstract/Free Full Text]
43. Nelissen-Vrancken H.J.M.G., Struijker-Boudier H.A.J., Daemen M.J.A.P., Smits J.F.M. Antihypertensive therapy and adaptive mechanisms in peripheral ischemia. Hypertension (1993) 22:780–788.[Abstract/Free Full Text]
44. Ziche M., Parenti A., Morbidelli L., Meiniger C.J., Granger H.J., Ledda F. The effect of vasoactive factors on the growth of coronary endothelial cells. Cardiologia (1992) 37:573–575.[Medline]
45. Gohlke P., Kuwer I., Schnell A., Amann K., Mall G., Unger T. Blockade of bradykinin B2 receptors prevents the increase in capillary density induced by chronic angiotensin- converting enzyme inhibitor treatment in stroke-prone spontaneously hypertensive rats. Hypertension (1997) 29:478–482.[Abstract/Free Full Text]
46. Sunderkotter C., Steinbrink K., Goebeler M., Bhardwaj J., Sorg C. Macrophages and angiogenesis. J Leukoc Biol (1994) 55:410–422.[Abstract]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				E. I. Dedkov, W. Zheng, and R. J. Tomanek Compensatory growth of coronary arterioles in postinfarcted heart: regional differences in DNA synthesis and growth factor/receptor expression patterns Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1686 - H1693. [Abstract] [Full Text] [PDF]

				B. Westendorp, R. G. Schoemaker, H. Buikema, F. Boomsma, D. J. van Veldhuisen, and W. H. van Gilst Progressive left ventricular hypertrophy after withdrawal of long-term ACE inhibition following experimental myocardial infarction Eur J Heart Fail, March 1, 2006; 8(2): 122 - 130. [Abstract] [Full Text] [PDF]

				B. Westendorp, R. G. Schoemaker, H. Buikema, D. de Zeeuw, F. Boomsma, W. H. van Gilst, and D. J. van Veldhuisen Beneficial effects of add-on hydrochlorothiazide in rats with myocardial infarction optimally treated with quinapril Eur J Heart Fail, December 1, 2005; 7(7): 1085 - 1094. [Abstract] [Full Text] [PDF]

				P. van der Meer, E. Lipsic, R. H. Henning, K. Boddeus, J. van der Velden, A. A. Voors, D. J. van Veldhuisen, W. H. van Gilst, and R. G. Schoemaker Erythropoietin Induces Neovascularization and Improves Cardiac Function in Rats With Heart Failure After Myocardial Infarction J. Am. Coll. Cardiol., July 5, 2005; 46(1): 125 - 133. [Abstract] [Full Text] [PDF]

				B. Westendorp, R. G Schoemaker, H. Buikema, D. de Zeeuw, D. J van Veldhuisen, and W. H van Gilst Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction Journal of Renin-Angiotensin-Aldosterone System, March 1, 2004; 5(1): 27 - 32. [Abstract] [PDF]

				R. Van Kerckhoven, R. van Veghel, P. R Saxena, and R. G Schoemaker Pharmacological therapy can increase capillary density in post-infarction remodeled rat hearts Cardiovasc Res, February 15, 2004; 61(3): 620 - 629. [Abstract] [Full Text] [PDF]

				R. A. de Boer, Y. M. Pinto, A. J.H. Suurmeijer, S. Pokharel, E. Scholtens, M. Humler, J. M. Saavedra, F. Boomsma, W. H. van Gilst, and D. J. van Veldhuisen Increased expression of cardiac angiotensin II type 1 (AT1) receptors decreases myocardial microvessel density after experimental myocardial infarction Cardiovasc Res, February 1, 2003; 57(2): 434 - 442. [Abstract] [Full Text] [PDF]

				M. Palmen, M.J.A.P. Daemen, R. Bronsaer, W.R.M. Dassen, H.R. Zandbergen, M. Kockx, J.F.M. Smits, R. van der Zee, and P.A. Doevendans Cardiac remodeling after myocardial infarction is impaired in IGF-1 deficient mice Cardiovasc Res, June 1, 2001; 50(3): 516 - 524. [Abstract] [Full Text] [PDF]

				R. Van Kerckhoven, E. A.J Kalkman, P. R Saxena, and R. G Schoemaker Altered cardiac collagen and associated changes in diastolic function of infarcted rat hearts Cardiovasc Res, May 1, 2000; 46(2): 316 - 323. [Abstract] [Full Text] [PDF]

				J. P.M Cleutjens, W.M. Blankesteijn, M. J.A.P Daemen, and J. F.M Smits The infarcted myocardium: Simply dead tissue, or a lively target for therapeutic interventions Cardiovasc Res, November 1, 1999; 44(2): 232 - 241. [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Nelissen-Vrancken, H.J.M. G. Articles by Smits, J. F.M. Search for Related Content PubMed PubMed Citation Articles by Nelissen-Vrancken, H.J.M. G. Articles by Smits, J. F.M. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics