AJP - Heart AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H1237-H1244, 2007. First published December 1, 2006; doi:10.1152/ajpheart.00965.2006 Free Article
0363-6135/07 $8.00
This Article
Free upon publication Free Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/H1237    most recent
00965.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rakusan, K.
Right arrow Articles by Ostadal, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rakusan, K.
Right arrow Articles by Ostadal, B.

CALL FOR PAPERS
Oxygen Sensing: Life and Death of a Cell

ANG II type 1 receptor antagonist irbesartan inhibits coronary angiogenesis stimulated by chronic intermittent hypoxia in neonatal rats

Karel Rakusan,1 Zuzana Chvojkova,2,3 Patricia Oliviero,2 Ivana Ostadalova,3 Frantisek Kolar,3 Catherine Chassagne,2 Jane-Lise Samuel,2 and Bohuslav Ostadal3

1Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada; 2Institut National de la Santé et de la Recherche Médicale Unité 572, Institut Fédératif de Recherche Jules Marrey, Paris, France; and 3Institute of Physiology, Academy of Sciences of the Czech Republic and Centre for Cardiovascular Research, Prague, Czech Republic

Submitted 6 September 2006 ; accepted in final form 27 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic hypoxia has been shown to stimulate myocardial microvascular growth and improve cardiac ischemic tolerance in young and adult rats. The aim of this study was to determine whether the ANG II type 1 receptor (AT1) pathway was involved in these processes. Newborn Wistar rats, exposed to chronic intermittent hypoxia (8 h/day) for 10 days, were simultaneously treated with AT1 receptor blocker irbesartan and compared with untreated animals. The major finding is that chronic hypoxia increased the capillary supply of myocardial tissue, which was even more pronounced in hypertrophied right ventricle, whereas increased arteriolar supply was found only in the left ventricle. This angiogenic response was completely prevented by irbesartan. Moreover, chronic hypoxia improved the postischemic recovery of cardiac contractile function during reperfusion, and this protective effect was also completely abolished by irbesartan. Chronic hypoxia increased the myocardial density of AT1 but not of ANG II type 2 receptor subtypes, whereas the effect of irbesartan was not significant. The expression of caveolin-1{alpha} markedly increased in response to chronic hypoxia, and irbesartan prevented this effect. Neither hypoxia nor irbesartan treatment altered the expression of nitric oxide synthase 3, heat shock protein 90, and VEGF. It is concluded that the AT1 receptor pathway plays an important role in coronary angiogenesis and improved cardiac ischemic tolerance induced in neonatal rats by chronic hypoxia.

angiotensin II receptors; ischemia-reperfusion; caveolin-1

ADAPTATION TO CHRONIC HYPOXIA increases cardiac tolerance to all major deleterious consequences of acute oxygen deprivation in both adult and immature heart. In addition to the protective effect, chronic hypoxia may also induce other adaptive responses, including hypoxic pulmonary hypertension and right ventricular (RV) hypertrophy (22). This is a beneficial adaptation, allowing the RV to cope with an increased afterload and to maintain cardiac output. Although many potential factors have been proposed to play a role in the mechanisms of cardiac adaptation to chronic hypoxia, the available data are not sufficiently conclusive. Moreover, a substantial amount of information concerning the adaptive response was obtained from the adult myocardium, whereas much less is known about this process in the immature heart. However, clinical relevance of the developmental approach is obvious: chronic hypoxia is the main pathophysiological feature of hypoxemic congenital heart disease. Understanding the mechanisms by which these malformations modify the myocardium and how they impact on the adaptive mechanisms during ischemia may provide insight into developing treatments for limiting myocardial damage during cardiac surgery.

One of the still unresolved problems in the process of cardiac adaptation to chronic hypoxia is the effect on coronary angiogenesis. The experimental data are inconsistent: some of them indicate increased, whereas others point to an unchanged or even decreased, ventricular capillary density (27, 29). Moreover, all morphometric studies were performed exclusively in the left ventricular (LV) myocardium of the adult animals; data on the RV in the immature heart are completely lacking. ANG II has been shown to play an important role in cardiac growth and coronary angiogenic responses to various stimuli in a number of experimental models. This effect is likely mediated by the ANG II type 1 (AT1) receptor pathway, although the available data are controversial (12, 36, 43). The role of ANG II receptors in normal and stimulated growth of coronary microvessels during early postnatal period has not been sufficiently examined (8).

The aim of the present study was to find out whether the AT1 receptor pathway is involved in the mechanisms of adaptive response of the neonatal heart to chronic hypoxia. Particular attention was paid to the effect on microvascular growth and cardiac tolerance to acute oxygen deprivation. The expression of specific proteins, such as caveolin-1, nitric oxide (NO) synthase 3 (NOS3), heat shock protein 90 (HSP90), and VEGF, was also analyzed to determine whether they are related to changes in coronary angiogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. Newborn Wistar rats of both sexes were obtained from the animal care facility of the Institute of Physiology in Prague. The day after birth, they were randomly distributed to mothers, and their number per litter was kept at 10 throughout the experiment. Chronic hypoxia was induced by exposures to intermittent high altitude (IHA) simulated in a hypobaric chamber. Barometric pressure was lowered to 405 mmHg (54 kPa), which corresponds to the altitude of 5,000 m (PO2 = 85 mmHg, 11.3 kPa). Mothers with offspring were exposed to hypoxia for 10 consecutive days, starting the next day after delivery; the duration of each daily exposure was 8 h. Normoxic animals were kept for the same period at barometric pressure and PO2 equivalent to the altitude of 200 m. Both hypoxic and normoxic groups contained four litters, and the offspring in each litter were randomly assigned to three subgroups: 1) untreated controls, 2) irbesartan treated, and 3) vehicle treated. Irbesartan (Bristol-Myers Squibb), dissolved in 0.8 vol% of 1 N sodium hydroxide, was administered to the offspring by a gavage in a dose of 40 µg/g body mass (BM) and volume of 10 µl/g BM daily before the hypoxic exposure. Another hypoxic and normoxic subgroup received the same volume of the vehicle. The animals were assigned to three series of experiments (heart function, morphometry, and biochemistry) and examined the next day after the last hypoxic exposure. No animal mortality was observed during the time course of the study. The experimental protocol was approved by the Animal Care and Use Committee of the Institute of Physiology, Academy of Sciences of the Czech Republic, and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

Morphometric analysis. The animals assigned for morphometric studies were decapitated, and their hearts were rapidly excised and washed in cold (5°C) saline. Subsequently, atrial tissue and large vessels were removed, and ventricles were fixed with 1.5% glutaraldehyde adjusted to pH 7.4 with phosphate buffer.

The histological methods have been described previously (13, 30). Briefly, after fixation and dehydration in alcohol, the samples were embedded in historesin. The middle portions of the free ventricular walls were used for morphometry. Sections (1 µm thick) were stained by Avallone's modification of the Jones silver methenamine method for staining basement membranes. This staining procedure gives a sharp black edge to basement membranes and, in this way, facilitates identification of capillaries and arterioles within tissue cross sections.

With the use of an image analyzer (Bioquant Meg V), the numbers of capillaries and arterioles per unit area of a cross section were determined, enabling us to calculate their respective numerical densities. Coronary arterioles were analyzed by using an image analyzer as follows. First, the total area of the cross section was measured. Afterward, all small arteries and arterioles were registered irrespective of the sectioning angle. For each arteriolar profile observed, the shortest (minimum) and longest (maximum) external diameters were measured together with the minimum internal diameter. These measurements enabled us to estimate the distribution of arterioles according to their size, to measure the arteriolar thickness, and to estimate the arteriolar length density (average length of arterioles per volume of tissue). Arteriolar length density was calculated on the basis of arteriolar numerical density and the ratios of the long to short axes from each arteriolar profile (3, 7).

In situ autoradiographic quantitative receptor binding assay. The animals assigned for biochemical studies were decapitated, and their hearts were rapidly excised and washed in cold (5°C) saline. Subsequently, atrial tissue and large vessels were removed, and ventricles were dissected into the free RV wall and the LV, including the septum. Both parts were weighed, and the LV was rapidly frozen in liquid nitrogen and then stored at –80°C until use.

Transverse heart cryosections (10 µm) were used for ANG II binding studies, which were performed as described earlier (20) with (3-[125I]iodotyrosyl-4,Sar1,Ile8)-ANG II (specific activity, 2,000 Ci/mmol; Amersham) in the presence or absence of losartan, PD-123319, or unlabeled ANG II. ANG II-specific total binding corresponds to the total (ANG II receptor) minus the nonspecific labeling revealed with unlabeled ANG II. AT1 and ANG II type 2 (AT2) receptor-specific bindings were measured in the presence of PD-123319 and losartan, respectively. The data (in fmol/mm2) are means of receptor binding capacity per surface unit.

Western blot analysis. Five ventricular cryostat sections (15 µm width) were lysed in boiling SDS buffer containing 1% SDS, 10 mmol/l Tris·HCl (pH 7.4), 1 mmol/l ortho-vanadate, 2 mg/ml leupeptine, 2 mg/ml aprotinine, and 10 mg/ml PMSF. Lysates were warmed in a microwave oven for 15 s (900 W) and subsequently clarified by centrifugation at 11,000 g for 15 min at 15°C. Supernatants were stored at –20°C. Lysate protein concentrations were measured with the use of a protein assay kit (Pierce) with BSA as a standard. The tissue contents of HSP90, caveolin-1{alpha}, NOS3, and VEGF were determined by Western blot analysis (28). Proteins (20 µg to detect caveolin-1 and caveolin-3; and 5 µg to detect caveolin-1{alpha}) were resolved by electrophoresis on 12% SDS-acrylamide gel and electrophoretically transferred to nitrocellulose membrane (Schleicher & Schuell) in 25 mM Tris, 192 mM glycin, 0.01% SDS, and 15% methanol. The membranes were blocked with Tris-buffered saline (TBS, pH 7.4), containing 0.1% Tween-20, 5% nonfat dry milk, and 1% BSA, for 1 h at room temperature (RT) before incubation for another 1 h at RT with either MAb anti-HSP90 (1:5,000, Transduction Laboratories), anti-NOS3 (SC-654, Santa Cruz), or with polyclonal antibody caveolin-1{alpha} (1:10,000, Santa Cruz) in TBS-Tween-20. After being washed, membranes were incubated for 1 h at RT with either anti-mouse IgG- or anti-rabbit IgG-conjugated to horseradish peroxidase (1:5,000, Amersham International). Ponceau S staining of each membrane confirmed that equal amounts of protein were loaded onto each lane. After being washed, immunoreactive bands were visualized by enhanced chemiluminescence, quantified by densitometry using a computer-based imaging system (Lass 1000, Fuji), and normalized to the actin amount present on the gel.

Isolated perfused heart. After the animal was euthanized by cervical dislocation, the chest was opened and a cannula was inserted into the aorta. The heart was rapidly excised, trimmed of atria, and perfused as described earlier (23) at a constant pressure (53 mmHg) with a nonrecirculating Krebs-Henseleit solution containing (in mmol/l) 118.0 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 KH2PO4, 7.0 glucose, and 1.1 mannitol. The solution was saturated by a mixture of 95% O2-5% CO2 (pH 7.4) and maintained at 37°C. The hearts were electrically stimulated at a rate of 200 beats/min throughout the experiment. The contractile function of the hearts was measured by using an isometric force transducer connected to the apex of the heart and analyzed by custom-designed software. The resting force was set to the level at which the developed force (DF) reached ~80% of the maximum force that was reached at optimum preload. Coronary flow was measured by timed collection of coronary effluent and normalized to cardiac mass. After a period of stabilization, the hearts were subjected to 40 min of global normothermic ischemia, followed by reperfusion until maximal recovery of DF was achieved. The values of postischemic recovery of DF were expressed as a percentage of preischemic values.

Statistics. Results are expressed as means ± SE. One-way ANOVA with subsequent Bonferroni post hoc tests was used for comparison of differences between groups. Differences were assumed as statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
BM and heart mass. BM and heart mass parameters of animals exposed to IHA hypoxia for 10 days after birth and their age-matched normoxic controls are summarized in Table 1. When compared with normoxic groups, IHA hypoxia led to a significant retardation of body growth accompanied by an increase of the RV mass, whereas the LV mass was unchanged. The RV mass normalized to BM was increased by 36% in hypoxic animals. Hypoxia also slightly but significantly elevated the level of hematocrit.


View this table:
[in this window]
[in a new window]

  		Table 1. Hematocrit, body mass, and heart mass parameters of control untreated, vehicle-treated, and irbesartan-treated rats exposed to IHA hypoxia and of age-matched normoxic animals

 
Treatment with irbesartan decreased BM as well as the RV and LV mass in both normoxic and hypoxic groups. After normalization of ventricular mass to BM, this inhibitory effect of irbesartan was statistically significant in hypoxic animals only. In contrast, irbesartan decreased hematocrit levels in normoxic but not in hypoxic groups. Animals treated with the vehicle did not differ from corresponding untreated subgroups in any parameter; therefore, only one control group was subjected to further analyses.

Morphometry of capillaries and arterioles. Exposure to IHA hypoxia resulted in a significant increase of capillary numerical density in the myocardial tissue, and this effect was even more pronounced in the hypertrophied RV. This angiogenic response was abolished by a simultaneous treatment with irbesartan (Fig. 1A). Increased arteriolar supply after exposure to hypoxia was found only in the LV, and it was also abolished by irbesartan (Fig. 1B). Morphometric analysis of coronary arterioles did not reveal any significant difference between untreated normoxic and hypoxic animals, except for thinning of the vessel wall in the latter group (Table 2). Irbesartan had no effect in the normoxic animals but significantly reduced arteriolar length density in the RV myocardium of the hypoxic group. Comparison of irbesartan-treated normoxic and hypoxic animals showed, in the latter group, 1) the decrease in arteriolar length density and internal diameter-to-thickness index, 2) the increase in both external and internal diameter and the ratio of capillaries to arterioles in the RV, and 3) the decrease in vessel thickness and internal diameter-to-thickness index in the LV (Table 2).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. Numerical density of capillaries (A) and arterioles (B) in the left and right ventricular myocardium of control untreated (white bars) and irbesartan-treated (black bars) rats exposed to intermittent high altitude (IHA) hypoxia and of age-matched normoxic animals. Values are means ± SE from 7–8 animals in each group. *P < 0.05 vs. corresponding normoxic group; +P < 0.05 vs. corresponding untreated group.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Morphometry of arterioles in the left and right ventricular myocardium of control untreated and irbesartan-treated rats exposed to IHA hypoxia and of age-matched normoxic animals

 
AT1 and AT2 receptor densities. The experimental approach used to quantify 125I-Sar1-Ile8-ANG II binding via AT1 and AT2 receptor subtype was validated previously (20). We analyzed the AT1 and AT2 binding capacities in the rat heart exposed to hypoxia with or without irbesartan treatment. 125I-Sar1-Ile8-ANG II was uniformly distributed throughout the heart. Preincubation with 10 µM of ANG II reduced binding in all structures (Fig. 2A). Preincubation with PD-123319 and losartan to detect specific binding to AT1 and AT2, respectively, also partially inhibited radioligand binding in cardiac tissues. Quantification of specific binding in the ventricles indicated that IHA hypoxia significantly increased the AT1 receptor subtype densities (Fig. 2B). Irbesartan treatment also tended to increase AT1 densities in both experimental conditions, but the effect did not reach statistical significance. It is worthy to note that the proportion of AT2 subtype was around 40% of the total ANG II receptors and did not vary among the experimental groups (Fig. 2C).


Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 2. 125I-Sar1-Ile8-ANG II binding in rat heart sections. Pseudocolor images of rat heart sections incubated in the presence of 125I-Sar1-Ile8-ANG II without or with preincubation in presence of ANG II are shown. PD-123319 or losartan reveals the total ANG II binding (ANG II-R), nonspecific binding (NS), and ANG II types 1 and 2 receptors (AT1 and AT2), respectively (A). Density of AT1 (B) and AT2 (C) receptors in untreated (white bars) and irbesartan-treated (black bars) rats exposed to IHA hypoxia and of age-matched normoxic animals. Note that chronic hypoxia increased only AT1 receptor density. Values are means ± SE from 8–12 hearts/group. C, normoxic; H, hypoxic; AU, arbitrary units. *P < 0.05 vs. corresponding normoxic group.

 
Expressions of NOS3, caveolin-1, HSP90, and VEGF. To determine whether the angiogenetic process was related to changes in specific changes in protein expression, we analysed the expression of caveolin-1{alpha} and NOS3, both of which are highly expressed in endothelial cells. The expression of caveolin-1 markedly increased in response to IHA hypoxia, and irbesartan treament prevented this increase (Fig. 3A). Neither IHA hypoxia nor irbesartan treament altered the expression of NOS3 and that of HSP90, an important regulator of NOS3 activity (Fig. 3, B and C). Finally, we analysed the expression of VEGF, a major growth factor involved in the angiogenetic process. Ten days after the induction of IHA hypoxia and/or irbesartan treatment, the level of VEGF expression was found to be similar to that of normoxic untreated animals (Fig. 3D).


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 3. Expression of caveolin-1{alpha} (Cav-1; A), nitric oxide synthase 3 (NOS3; B), heat shock protein 90 (HSP90; C), and VEGF (D) in left ventricular myocardium of untreated (white bars) and irbesartan-treated (black bars) rats exposed to IHA hypoxia and of age-matched normoxic animals evaluated by quantitative Western blot analysis. CI, normoxic, irbesartan-treated; HI, hypoxic; irbesartan-treated. Values are means ± SE from 6–8 hearts/group. *P < 0.05 vs. corresponding normoxic group; +P < 0.05 vs. corresponding untreated group.

 
Cardiac ischemic tolerance. Baseline preischemic DF of the isolated perfused heart, measured at the same preload and normalized to heart mass, did not differ between untreated normoxic and hypoxic groups. Similarly, chronic hypoxia had no effect on specific coronary flow both during the preischemic period and during reperfusion after the period of global ischemia. Irbesartan treatment moderately increased baseline DF in both groups, baseline coronary flow in the normoxic group, and the recovery of flow during reperfusion in the hypoxic group, compared with corresponding untreated groups (Table 3). Chronic hypoxia significantly improved the postischemic recovery of contractile function during reperfusion, and this protective effect was completely abolished by treatment with irbesartan (Fig. 4).


View this table:
[in this window]
[in a new window]

  		Table 3. Developed force and coronary flow of isolated perfused hearts of control untreated and irbesartan-treated rats exposed to IHA hypoxia and of age-matched normoxic animals

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Cardiac tolerance to ischemia expressed as recovery of developed force (DF). Maximal postischemic recovery of DF measured in isolated perfused hearts of control untreated (white bars) and irbesartan-treated (black bars) rats exposed to IHA hypoxia and of age-matched normoxic animals. Values are means ± SE from 9–10 hearts/group. *P < 0.05 vs. corresponding normoxic group; +P < 0.05 vs. corresponding untreated group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present data demonstrate that chronic intermittent hypoxia in neonatal rats induced an angiogenetic process that was associated with increases in caveolin-1 expression and in AT1 receptor density. Chronic treatment with the AT1 receptor blocker irbesartan prevented these responses. These effects occured in addition to the classical adaptative responses such as erythropoiesis or RV hypertrophy.

Animals exposed to chronic intermittent hypoxia, corresponding to 5,000 m during the first 10 days of postnatal life, exhibited body growth retardation, RV hypertrophy, and polycythemia. The extent of these adaptive responses to hypoxia was comparable with those of our previous studies using the same experimental model (23). Chronic treatment with AT1 receptor blocker irbesartan during the same developmental period led to a proportional retardation of body and heart growths since the normalized mass of the two ventricles was not appreciably affected in the normoxic group. On the other hand, irbesartan decreased the LV-to-BM ratio in hypoxic animals and completely prevented hypoxia-induced RV hypertrophy. It is unlikely that this effect would be due to altered sensing of hypoxia because AT1 blockade had no effect on the increase in hematocrit in the hypoxic group. These results suggest that ANG II through its AT1 receptors plays an important role in the growth response of the immature heart to hypoxia. This hypothesis is strengthened by the AT1 receptor density, which was found to be increased in immature hearts submitted to hypoxia, whereas the AT2 receptor subtype was not affected.

The major finding of our study is that chronic intermittent hypoxia of a relatively short duration stimulated angiogenesis in developing hearts and that treatment with the AT1 blocker completely prevented this response. Early postnatal ontogeny is associated with rapid growth of the myocardial microvascular bed (18, 21, 27). Chronic hypoxia further stimulated this process as evident from markedly increased arteriolar density in the LV and capillary densities in both LV and RV. The increase of capillary formation was actually even more pronounced in the RV, which hypertrophied due to hypoxic pulmonary hypertension. These data are in line with previous reports indicating that chronic hypoxia may increase capillary density in the myocardium of young and adult animals (25) (for reviews, see Refs. 28 and 40). However, to our knowledge, the present study is the first one that analyzed microvascular morphometry in the RV myocardium of neonatal rats. The process of angiogenesis, as evidenced by morphometric analysis, is strengthened by biochemical data. Indeed, caveolin-1 level, a good marker of capillarization, increased parallel to the vessel density. Conversely, the levels of NOS3, as well as of its molecular partner HSP90, were not altered by hypoxia at this stage, indicating that NO pathway is unlikely to play a pivotal role in this model.

Hypoxia also induced an increased density of AT1 receptors, whereas the binding capacity of AT2 remained unaltered. This is in line with previous data in adults (1), albeit the time course differed. The increased expression of AT1 receptors in the myocardium of chronically hypoxic rats and the absence of an angiogenic response in irbesartan-treated animals suggest that ANG II plays a major role in hypoxia-induced formation of new arterioles and capillary sprouting by acting on AT1 receptors. This observation is in line with a number of reports demonstrating that proangiogenic effects of ANG II are mediated by AT1 receptors in various in vivo and in vitro experimental models. Thus long-term AT1 blockade decreased densities of subepicardial arterioles in spontaneous hypertensive rats (26) and capillaries in hearts of cardiomyopathic hamsters (34) or in rats with noninsulin-dependent diabetes (14). AT1 blockade also had inhibitory effects in various models of tumor-associated angiogenesis (11, 38, 42) and in the growth of microvessels induced by ANG II (19), aldosterone (17), electrical stimulation (2), or ischemia (32) in skeletal muscle. Further evidence for a proangiogenic effect of AT1 receptor stimulation comes from studies on AT1-receptor knockout mice, which exhibit impaired growth of microvessels in tumors (10) and border zone of infarcted myocardium (39). It appears that AT1-dependent angiogenic effects of ANG II involve activation of growth factors, such as VEGF, and inflammatory pathways (2, 10, 32, 35, 38). In our study, however, myocardial VEGF protein level was appreciably modified by neither chronic hypoxia nor irbesartan treatment. In agreement, previous studies including ours on adult rats demonstrated only a transient increase in VEGF transcript at the onset of hypoxic exposure (9, 25).

Although AT2 receptor density was not altered in our experiments, we cannot exclude that this subtype was involved in the inhibition of angiogenesis by irbesartan. The occurence of AT2 receptors in the neonatal myocardium is higher than in adulthood (20), and AT2 signaling pathway could be "unmasked" because of higher availability of ANG II when AT1 receptors are blocked. Moreover, the antiangiogenic effect of AT2 stimulation has been postulated in several studies (5, 6, 19, 36). However, the role of the two ANG II receptor subtypes in microvascular growth remains rather controversial. In contrast to the above-mentioned studies, AT1 blockade was shown to increase capillary density in rat hearts subjected to chronic myocardial infarction (33, 37), as well as in hearts of stroke-prone spontaneously hypertensive rats (44). In rats overexpressing the AT1 receptor, myocardial infarction decreased microvessel density, and this was amenable to AT1 blockade (8). Recently, Walther et al. (43) demonstrated AT1-mediated inhibitory effect of ANG II on tumor-associated angiogenesis, whereas AT2 receptors exerted opposite, proangiogenic action. The reason for these conflicting results concerning the role of ANG II and its two receptors pathways in various in vivo and in vitro models of stimulated angiogenesis remains to be elucidated.

The present study clearly indicates that coronary flow does not reflect a variation in capillary density. Values of specific flow did not differ between normoxic and hypoxic hearts, although both capillary and arteriolar densities were increased in the latter group. Moreover, irbesartan abolished the hypoxia-stimulated angiogenesis but increased the coronary flow. Apparently, coronary flow measured under these conditions reflects differences in the control of vascular tone in various groups of hearts rather than differences in coronary morphometry. The fact that the expression of both NOS3 and HSP90 was unaffected by chronic hypoxia is in agreement with the fact that the coronary flow is dependent on humoral factors (e.g., adenosine, NO, endothelin, and ANG II).

The improved recovery of cardiac contractile function in the hypoxic group confirms our previous observations (23), as well as those of others (4, 41), indicating that the already high ischemic tolerance of the immature heart can be further increased by chronic hypoxia. Despite a certain recent progress in understanding this protective phenomenon, its detailed mechanism is still far from clear (15). Obviously, it involves complex adaptive changes of cardiomyocytes that increase their ability to cope with acute oxygen deprivation. Increased capillarization of the myocardium plays an important role in improving geometrical conditions for oxygen diffusion during exposure to chronic hypoxia, but it can hardly contribute to the improved cardiac tolerance in the setting of acute no-flow ischemia. This view is supported by observations that the improved tolerance of chronically hypoxic hearts to acute oxygen deprivation is manifested even in nonperfused myocardial preparations (16).

A novel finding of the present study is that irbesartan completely blocks the cardioprotective effect of chronic hypoxia, suggesting that activation of AT1 receptors is important for its manifestation. In this connection it is interesting to mention the results of Oudot et al. (24) that angiotensin AT1 receptor stimulation would exert protective properties during myocardial ischemia-reperfusion. The fact that the postischemic recovery of contractile function was low despite the highest recovery of flow in the irbesartan-treated hypoxic group is in line with the view that the inhibitory effect of AT1 blockade on cardiac ischemic tolerance takes place at the level of myocytes rather than microcirculation.

It may be concluded that AT1 is involved in the mechanisms of cardiac adaptation to chronic hypoxia, at least in neonatal rats. The blockade of AT1 receptors by irbesartan completely abolished not only the angiogenic response to chronic hypoxia but also its cardioprotective effect. The involvement of AT1 receptor pathway in the adaptive responses of immature hearts to hypoxia should be taken into consideration in the treatment of children suffering from cyanotic congenital heart disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from Bristol-Myers Squibb, Institut National de la Santé et de la Recherche Médicale, Fondation de France, Ministry of Education of the Czech Republic (1M0J10).


   ACKNOWLEDGMENTS
 
We thank Ching Kuo and Sylvie Lachapelle for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Ostadal, Inst. of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic (e-mail: ostadal{at}biomed.cas.cz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamy C, Oliviero P, Eddahibi S, Rappaport L, Samuel JL, Teiger E, Chassagne C. Cardiac modulations of ANG II receptor expression in rats with hypoxic pulmonary hypertension. Am J Physiol Heart Circ Physiol 283: H733–H740, 2002.[Abstract/Free Full Text]
  2. Amaral SL, Linderman JR, Morse MM, Greene AS. Angiogenesis induced by electrical stimulation is mediated by angiotensin II and VEGF. Microcirculation 8: 57–67, 2001.[CrossRef][Web of Science][Medline]
  3. Anversa P, Capasso JM. Loss of intermediate-sized coronary arteries and capillary proliferation after left ventricular failure in rats. Am J Physiol Heart Circ Physiol 260: H1552–H1560, 1991.[Abstract/Free Full Text]
  4. Baker EJ, Boerboom LE, Olinger GN, Baker JE. Tolerance of the developing heart to ischemia: impact of hypoxemia from birth. Am J Physiol Heart Circ Physiol 268: H1165–H1173, 1995.[Abstract/Free Full Text]
  5. Benndorf R, Boger RH, Ergun S, Steenpass A, Wieland T. Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells. Circ Res 93: 438–447, 2003.[Abstract/Free Full Text]
  6. Brogelli L, Parenti A, Ledda F. Inhibition of vascular smooth muscle cell growth by angiotensin type 2 receptor stimulation for in vitro organ culture model. J Cardiovasc Pharmacol 39: 739–745, 2002.[CrossRef][Web of Science][Medline]
  7. Chen Y, Torry RJ, Baumbach GL, Tomanek RJ. Proportional arteriolar growth accompanies cardiac hypertrophy induced by volume overload. Am J Physiol Heart Circ Physiol 267: H2132–H2137, 1994.[Abstract/Free Full Text]
  8. De Boer RA, Pinto YM, Suurmeijer AJ, Pokharel S, Scholtens E, Humler M, Saavedra JM, Boomsma F, van Gilst WH, van Veldhuisen DJ. Increased expression of cardiac angiotensin II type 1 (AT1) receptors decreases myocardial microvessel density after experimental myocardial infarction. Cardiovasc Res 57: 434–442, 2003.[Abstract/Free Full Text]
  9. Deindl E, Kolar F, Neubauer E, Vogel S, Schaper W, Ostadal B. Effect of intermittent high altitude hypoxia on gene expression in rat heart and lung. Physiol Res 52: 147–157, 2003.[Web of Science][Medline]
  10. Egami K, Murohara T, Shimada T, Sasaki K, Shintani S, Sugaya T, Ishii M, Akagi T, Ikeda H, Matsuishi T, Imaizumi T. Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth. J Clin Invest 112: 67–75, 2003.[CrossRef][Web of Science][Medline]
  11. Fujita M, Hayashi I, Yamashina S, Itoman M, Majima M. Blockade of angiotensin AT1a receptor signaling reduces tumor growth, angiogenesis, and metastasis. Biochem Biophys Res Commun 294: 441–447, 2002.[CrossRef][Web of Science][Medline]
  12. Greene AS, Amaral SL. Microvascular angiogenesis and the renin-angiotensin system. Curr Hypertens Rep 4: 56–62, 2002.[Web of Science][Medline]
  13. Heron MI, Rakusan K. Short- and long-term effects of neonatal hypo- and hyperthyroidism on coronary arterioles in rat. Am J Physiol Heart Circ Physiol 271: H1746–H1754, 1996.[Abstract/Free Full Text]
  14. Jesmin S, Hattori Y, Sakuma I, Mowa CN, Kitabatake A. Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model. Am J Physiol Heart Circ Physiol 283: H1387–H1397, 2002.[Abstract/Free Full Text]
  15. Kolar F, Ostadal B. Molecular mechanisms of cardiac protection by adaptation to chronic hypoxia. Physiol Res 53, Suppl 1: S3–S13, 2004.
  16. McGrath JJ, Prochazka J, Pelouch V, Ostadal B. Physiological responses of rats to intermittent high-altitude stress: effects of age. J Appl Physiol 34: 289–293, 1973.[Free Full Text]
  17. Michel F, Ambroisine ML, Duriez M, Delcayre C, Levy BI, Silvestre JS. Aldosterone enhances ischemia-induced neovascularization through angiotensin II-dependent pathway. Circulation 109: 1933–1937, 2004.
  18. Muhlfeld C, Singer D, Engelhardt N, Richter J, Schmiedl A. Electron microscopy and microcalorimetry of the postnatal rat heart (Rattus norvegicus). Comp Biochem Physiol A Mol Integr Physiol 141: 310–318, 2005.[CrossRef][Medline]
  19. Munzenmaier DH, Greene AS. Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension 27: 760–765, 1996.[Abstract/Free Full Text]
  20. Oliviero P, Chassagne C, Kolar F, Adamy C, Marotte F, Samuel JL, Rappaport L, Ostadal B. Effect of pressure overload on angiotensin receptor expression in the rat heart during early postnatal life. J Mol Cell Cardiol 32: 1631–1645, 2000.[CrossRef][Web of Science][Medline]
  21. Ostadal B, Ostadalova I, Dhalla NS. Development of cardiac sensitivity to oxygen deficiency: comparative and ontogenetic aspects. Physiol Rev 79: 635–659, 1999.[Abstract/Free Full Text]
  22. Ostadal B, Ostadalova I, Kolar F, Pelouch V, Dhalla NS. Cardiac adaptation to chronic hypoxia. Adv Org Biol 6: 43–90, 1998.
  23. Ostadalova I, Ostadal B, Jarkovska D, Kolar F. Ischemic preconditioning in chronically hypoxic neonatal rat heart. Pediatr Res 52: 561–567, 2002.[CrossRef][Web of Science][Medline]
  24. Oudot A, Vergely C, Ecarnot-Laubriet A, Rochette L. Angiotensin II activates NADPH oxidase in isolated rat hearts subjected to ischemia-reperfusion. Eur J Pharmacol 462: 145–154, 2003.[CrossRef][Web of Science][Medline]
  25. Partovian C, Adnot S, Eddahibi S, Teiger E, Levame M, Dreyfus P, Raffestin B, Frelin C. Heart and lung VEGF mRNA expression in rats with monocrotaline- or hypoxia-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 275: H1948–H1956, 1998.[Abstract/Free Full Text]
  26. Pu Q, Larouche I, Schiffrin EL. Effect of dual angiotensin converting enzyme/neutral endopeptidase inhibition, angiotensin converting enzyme inhibition, or AT1 antagonism on coronary microvasculature in spontaneously hypertensive rats. Am J Hypertens 16: 931–937, 2003.[CrossRef][Web of Science][Medline]
  27. Rakusan K. Vascularization of the heart during normal and pathological growth. Adv Org Biol 7: 130–153, 1999.
  28. Rakusan K, Cicutti N. The effect of acute and chronic hypoxia on coronary microcirculation. Hypoxia Med J 3: 3–7, 1996.
  29. Rakusan K, Cicutti N, Sladek T. Ischemic heart: microcirculatory aspects. Adv Exp Med Biol 388: 457–461, 1996.[Medline]
  30. Rakusan K, Cicutti N, Kazda S, Turek Z. Effect of nifedipine on coronary capillary geometry in normotensive and hypertensive rats. Hypertension 24: 205–211, 1994.[Abstract/Free Full Text]
  31. Ratajczak P, Damy T, Heymes C, Oliviero P, Marotte F, Robidel E, Sercombe R, Boczkowski J, Rappaport L, Samuel JL. Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat. Cardiovasc Res 57: 358–369, 2003.[Abstract/Free Full Text]
  32. Sasaki K, Murohara T, Ikeda H, Sugaya T, Shimada T, Shintani S, Imaizumi T. Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis. J Clin Invest 109: 603–611, 2002.[CrossRef][Web of Science][Medline]
  33. Schieffer B, Wirger A, Meybrunn M, Seitz S, Holtz J, Riede UN, Drexler H. Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat. Circulation 89: 2273–2282, 1994.
  34. Shimizu T, Okamoto H, Chiba S, Matsui Y, Sugawara T, Akino M, Nan J, Kumamoto H, Onozuka H, Mikami T, Kitabatake A. VEGF-mediated angiogenesis is impaired by angiotensin type 1 receptor blockade in cardiomyopathic hamster hearts. Cardiovasc Res 58: 203–212, 2003.[Abstract/Free Full Text]
  35. Siddiqui AJ, Mansson-Broberg A, Gustafsson T, Grinnemo KH, Dellgren G, Hao X, Fischer H, Sylven C. Antagonism of the renin-angiotensin system can counteract cardiac angiogenic vascular endothelial growth factor gene therapy and myocardial angiogenesis in the normal heart. Am J Hypertens 18: 1347–1352, 2005.[CrossRef][Web of Science][Medline]
  36. Silvestre JS, Tamarat R, Senbonmatsu T, Icchiki T, Ebrahimian T, Iglarz M, Besnard S, Duriez M, Inagami T, Levy BI. Antiangiogenic effect of angiotensin II type 2 receptor in ischemia-induced angiogenesis in mice hindlimb. Circ Res 90: 1072–1079, 2002.[Abstract/Free Full Text]
  37. Sladek T, Sladkova J, Kolar F, Papousek F, Cicutti N, Korecky B, Rakusan K. The effect of AT1 receptor antagonist on chronic cardiac response to coronary artery ligation in rats. Cardiovasc Res 31: 568–576, 1996.[CrossRef][Web of Science][Medline]
  38. Tamarat R, Silvestre JS, Durie M, Levy BI. Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways. Lab Invest 82: 747–756, 2002.[Web of Science][Medline]
  39. Toko H, Zou Y, Minamino T, Masaya M, Harada M, Nagai T, Sugaya T, Terasaki F, Kitaura Y, Komuro I. Angiotensin II type 1a receptor is involved in cell infiltration, cytokine production, and neovascularization in infarcted myocardium. Arterioscler Thromb Vasc Biol 24: 664–670, 2004.[Abstract/Free Full Text]
  40. Tomanek RJ, Lund DD, Yue X. Hypoxic induction of myocardial vascularization during development. Adv Exp Med Biol 543: 139–149, 2003.[Web of Science][Medline]
  41. Uy RG, Ross-Ascuitto NT, Ascuitto RJ. Recovery of the chronically hypoxic young rabbit heart reperfused following no-flow ischemia. Pediatr Cardiol 27: 37–46, 2006.[CrossRef][Web of Science][Medline]
  42. Walsh DA, Hu DE, Wharton J, Catravas JD, Blake DR, Fan TP. Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma. Br J Pharmacol 120: 1302–1311, 1997.[CrossRef][Web of Science][Medline]
  43. Walther T, Menrad A, Orzechowski HD, Siemeister G, Paul M, Schirner M. Differential regulation of in vivo angiogenesis by angiotensin II receptors. FASEB J 17: 2061–2067, 2003.[Abstract/Free Full Text]
  44. Xie Z, Gao M, Togashi H, Saito H, Koyama T. Improvement in the capillarity of the left ventricular wall of stroke-prone spontaneously hypertensive rats following angiotensin II receptor blockade. Clin Exp Hypertens 21: 441–452, 1999.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. P. Christensen, R.-l. Zhang, W. Zheng, J. J. Campanelli, E. I. Dedkov, R. M. Weiss, and R. J. Tomanek
Postmyocardial infarction remodeling and coronary reserve: effects of ivabradine and beta blockade therapy
Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H322 - H330.
[Abstract] [Full Text] [PDF]

This Article
Free upon publication Free Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/H1237    most recent
00965.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rakusan, K.
Right arrow Articles by Ostadal, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rakusan, K.
Right arrow Articles by Ostadal, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.