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From U127 INSERM (A.S., J.-L.S., F.M., L.R.) and U141 INSERM (P.P.,
B.I.L.), IFR Circulation Lariboisière, Paris, France.
Correspondence to Dr Jane-Lyse Samuel, INSERM U127, IFR Circulation, Hôpital Lariboisière, Université D. Diderot, 41 Blvd de la Chapelle, 75010 Paris, France.
Abstract
Abstract—The long-lasting effect of
angiotensin II (Ang II) on the microvasculature in the rat
left ventricle was studied. Immunolabeling of ventricular
cryosections combined with morphometric analysis allowed us to
(1) distinguish between capillaries and arterioles and (2) precisely
evaluate their respective densities in the endomyocardium.
Ang II–induced hypertensive cardiac hypertrophy was
associated with an 18% decrease in capillary density
(P<0.05) and an increase in arteriole density (+54%,
P<0.001). Treatments with losartan or PD123319,
the respective antagonists of the angiotensin
subtype 1 and subtype 2 receptors, prevented the increase in arteriolar
density, whereas only losartan, which restored normal
arterial pressure, prevented changes in capillary density.
Taken together, these results indicate that Ang II–induced cardiac
hypertrophy was associated with capillary rarefaction and
arteriolar growth, the 2 processes being independently regulated.
It has been widely
shown in both clinical and experimental investigations that
vulnerability to hypoxia is greater in the
endomyocardium of the left ventricle than in the epicardial
layers.1 2 This phenomenon is amplified in
hypertension-induced cardiac hypertrophy because the left
ventricular hypertrophy is accompanied by
structural rarefaction of capillaries, particularly in the
endomyocardium.3 4 The capillary
rarefaction is presumed to be secondary to the dominant myocyte
hypertrophy, but little is known about the cardiac
angiogenesis and/or vasculogenesis that may occur. There is evidence
that Ang II, the main effector peptide of the
renin-angiotensin system, may act as an angiogenic growth
factor in various tissues such as the rabbit cornea, the chick
chorioallantoic membrane,5 and the skeletal
muscle,6 whereas chronic treatment with an ACE
inhibitor reduces microvascular growth and even induces
arteriolar rarefaction.7
AT2, one of the Ang II receptor
subtypes,8 has been suggested to play a major
role in Ang II–induced angiogenesis.9
Surprisingly, in the hearts of genetically hypertensive rats
(spontaneously hypertensive rats), ACE inhibitor has been
reported to increase capillary density independently of its
antihypertensive and antihypertrophic effects.10
The apparent discrepancies relative to the angiogenic effect of Ang II
in skeletal and cardiac muscles might reflect tissue specificity,
differences in experimental models, or the type of vessels
analyzed. Therefore, the major aim of this study was to
investigate the effects of hypertension secondary to chronic
administration of Ang II on the density of coronary arterioles
and capillaries in rats. On the basis that the cellular responses to
Ang II are mediated by at least 2 receptor subtypes,
AT1 and
AT2,8 we analyzed
the effect of their respective antagonists losartan
and PD123319.
Methods
Animals
The rats were anesthetized with sodium pentobarbital. The
hearts, arrested in diastole (intravenous
injection of saturated solution of KCl), were quickly removed, weighed,
and cut transversally at the equator of the ventricles. The upper parts
of the hearts were mounted, frozen in isopentane precooled with liquid
nitrogen, and kept at -70°C until use.
Antibodies and Immunolabeling
Morphometric Analysis
Statistical Analysis
Results
Three-week Ang II–infusion increased rat systolic blood
pressure (P<0.01 versus control) and ratio of heart weight
to body weight, the index of cardiac hypertrophy
(Table
Characterization of Microvasculature in Endomyocardium
of Rat Left Ventricle
Effects of Ang II on Endomyocardial Microvasculature
Arteriolar Density
Discussion
The present study analyzed the Ang II–dependent
changes in arterial pressure and
endomyocardial microvasculature occurring in the
rat.
An immunohistochemical method capable of identifying arterioles within
microvasculature by distinguishing the VSMC differentiation state has
been tested previously.17 On the other hand, it
has been established that pericytes of true capillaries
(mid-capillaries) lack the SM
Changes in Arteriolar Density
Changes in Capillary Density
Conclusions
Selected Abbreviations and Acronyms
Acknowledgments
This work was supported by INSERM, CNRS, European Union
(Biomed), and Fondation de France.
Received January 24, 1998;
first decision February 10, 1998;
accepted April 17, 1998.
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© 1998 American Heart Association, Inc.
Third Workshop on Structure and Function of Large
Arteries: Part II
Microvasculature in Angiotensin II–Dependent Cardiac Hypertrophy in the Rat
Key Words: arterioles • capillaries • hypertrophy • angiotensin II
A total of 28 male normotensive Wistar rats weighing 300±20 g
were used in this study and belong to the series previously
described.11 12 The rats in this series were used
to demonstrate AT1- and
AT2-mediated changes in the coronary
artery walls.12 Osmotic minipumps (Alzet, model 2
ML4; constant rate of 2.5 µL/h) were filled with solvent vehicle
(n=9) or Ang II (n=7; 120 ng · kg-1
· min-1; Sigma) and implanted in rats. A third
group (n=6) simultaneously received Ang II and PD123319 (30
mg · kg-1 ·
d-1) in 2 minipumps, and a fourth group of rats
infused with Ang II (n=6) received losartan (10 mg ·
kg-1 · d-1) for 22
days in drinking water. All rats were treated for 23 days; at the end
of the treatment period, systolic blood pressure was measured
by the tail-cuff method.
The antibodies used were monoclonal antibodies directed against
human SM 
-actin (Dako) or SM -actinin (Sigma), rabbit polyclonal
antibodies directed against total fibronectin, laminin (Chemicon), or
vWf (Dako). Serial ventricular cryosections (5 µm)
were labeled using a double immunolabeling technique previously
described in detail.12 13 Briefly, sections were
successively incubated with antibodies directed against either (1) SM
-actin or SM -actinin (dilution 1/50 or 1/100, respectively) or
(2) total fibronectin (1/100), laminin (1/100), or vWf (1/200).
Afterward, sections were treated with biotinylated anti-mouse IgG
(1/200, Vector Laboratories), with FITC anti-rabbit IgG (Amersham), and
then with a streptavidin–Texas Red complex (1/50, Amersham). Sections
were mounted in aqueous medium (Fluoprep, Biomerieux) and observed
using a Leica microscope.
In every heart, analysis of the arterioles and capillary
density, respectively, was performed in 3 nonconsecutive serial
sections of the median part of the myocardium using a video
imaging microscopy approach.13 Vessel density was
evaluated throughout the inner third (endomyocardium) of
the entire left ventricle (septum and free wall). For each section, 25
to 35 fields (magnification x100) were recorded to perform the
analysis of the entire left ventricle circumference. Evaluation
of capillary density was performed in cardiac sections labeled with
total fibronectin antibody14 and based on the
quantification of positively labeled structures with a circular shape
and <8 µm lumen size. The arterioles were analyzed in
sections that were double immunolabeled for SM -actin and vWf. Only
the small blood vessels (lumen diameter <20 µm) positively
stained with vWf antibodies, surrounded by 1 SM layer (stained with
anti–SM -actin antibody), and oriented transversally were
considered.
Data per heart were the means of the quantification from 3
sections. All the quantitative analyses were performed in a
blinded fashion by 2 independent observers. Data are expressed as
mean±SEM. Statistical significance was assessed using 1-way ANOVA
followed by Scheffé's test. P<0.05 was considered
significant. 
). Losartan but not
PD123319 treatment prevented these Ang II–induced changes in
arterial pressure and heart weight to body weight
ratio.
View this table:
[in a new window]
Table 1. Index of Hypertension and Cardiac Hypertrophy
The specific double immunolabelings used allowed the
characterization of 2 types of small vessels (Figure 1). The vWf antibodies revealed the
endothelial layer of all vascular beds, whereas laminin
and fibronectin staining underlined both the cardiomyocytes
and all the blood vessels. The specific immunolabeling pattern for SM
cell marker permitted us to discriminate the capillaries, which were
negative for SM -actin and SM -actinin, from other vessels
present in the endomyocardium that stained positively
for these SM markers. These vessels were considered as arterioles on
the basis of their circular shape, their lumen size, which was slightly
larger than that of capillaries (lumen diameter of 10 to 20 µm
and 6 to 8 µm, respectively), and especially the presence of 1
layer of SM cells.
View larger version (74K):
[in a new window]
Figure 1. Phenotype of myocardial arterioles. Left
ventricular cryosections were double immunolabeled with
antibodies directed against total fibronectin and SM -actin (A) or
with vWf and SM -actinin (B). Cells of the arteriolar wall (arrows)
that expressed SM -actin or SM -actinin and are surrounded by
basal lamina exhibit a mature SM cell phenotype.
Capillary Density and Cardiac Hypertrophy
The capillary density in controls was 2775.44±78 capillaries per
square millimeter. A regression analysis of the animal
population, independent of the experimental groups, indicated a
negative linear correlation between the capillary density and the ratio
of heart weight to body weight (r2=0.52,
P=0.0011), confirming that in the heart the changes in
capillary density are correlated mainly to the degree of cardiac
hypertrophy.15 16 Three weeks of Ang
II infusion induced a 18±2% decrease in capillary density compared
with control (P<0.05 versus control) (Figure 2). Losartan but not PD123319
treatment prevented the Ang II–induced changes in capillary
density.
View larger version (83K):
[in a new window]
Figure 2. Mean density of coronary capillaries in
the endomyocardium of the left ventricle in different
experimental groups. Note that the Ang II–induced decrease in
capillary density was prevented by losartan (Los) but not by
PD123319 (PD) treatment. Values are mean±SEM. **P<0.05
vs control. C indicates control.
In the endomyocardium of the left ventricle, the
number of arterioles positively stained with both SM -actin and vWf
antibodies increased in the Ang II–treated group compared with control
(Figure 3A). The frequency histogram of
the arteriole density exhibited a gaussian distribution in both normal
and hypertensive rat hearts, but the histogram of Ang II–treated
animals was significantly shifted toward high vascular densities
(P<0.01 versus control) (Figure 3B). Because the histograms
showed a gaussian distribution for every group studied (data not
shown), the average values were considered further. Figure 3C shows
that the average arteriole density in the endomyocardium
area was significantly increased in the Ang II–treated group only
(P<0.001 versus other groups).
View larger version (91K):
[in a new window]
Figure 3. A, Distribution of coronary
arterioles throughout the endomyocardium of control (a, b)
or Ang II–treated (c, d) rats double immunolabeled with antibodies
directed against SM -actin (a, c) and vWf (b, d). In panel b, arrows
indicate some of the vessels that positively stained with SM
-antibodies. Note that the density of vessels positively
stained with SM -actin increased after treatment with Ang II when
compared with controls. Bar=50 µm. B, Frequency histograms of
arteriolar density in the endomyocardial left
ventricle of control (open columns) and Ang II–treated (solid columns)
rats. The shift to the right of the frequency histogram in the Ang
II–treated group indicates a homogeneous increase in
arteriolar density. C, Mean density of arterioles in the
endomyocardium of the left ventricle from different
experimental groups. The increased arteriole density is observed in the
Ang II–treated rats. Data are the mean±SEM of 6 individual rat
values. **P<0.001 vs control. C indicates control; Los,
losartan; and PD, PD123319.-actin.18
Therefore, to investigate whether Ang II alters the microvasculature of
the heart as suggested by others,7 we used an
immunocytochemical technique with a large set of antibodies to
distinguish between true capillaries and arterioles (Figure 1
). When
combined with a morphometric approach, it was possible to determine
capillary and arteriole densities. The capillary density found in
normal rat hearts was similar to those previously measured using
different experimental approaches.15 16 On the
other hand, the density of arterioles was 2- to 3-fold higher than that
already published.19 It is noteworthy that under
our conditions both arterioles and precapillary microvascular segments
were taken into account. Therefore, our experimental approach is a
sensitive method that constitutes an useful tool to investigate the
microvasculature in the rat heart during pathological conditions.
The present study provides strong evidence that Ang II induces
an increased arteriolar density in the
endomyocardial layer as reflected by the increased
density of vascular structures, with VSMCs exhibiting a contractile
phenotype (expressing SM -actin and SM -actinin). As
proposed by Price et al,17 the increase in the
arteriolar density might be due to an arterialization of
preexisting capillaries. Therefore, the perivascular cells exhibiting a
VSMC phenotype could result from the migration of dividing
VSMCs initially located in the upstream terminal arterioles.
Alternatively, they may derive from pericytes as described in the lung
capillaries of hypertensive animals.20 It has
been suggested that the qualitative and quantitative changes described
here are triggered by both mechanical17 21 and
humoral factors. The comparative analysis of Ang II
hypertensive rats treated with specific antagonists of
AT1 and AT2 receptors was
undertaken to discriminate between vascular effects directly dependent
on Ang II receptor subtypes from those dependent on pressure. Under our
experimental conditions, it was not possible to distinguish between
pressor mechanisms and the direct involvement of
AT1 receptor subtype, since losartan was
able to normalize both arterial pressure and arteriolar
density. Interestingly, PD123319 treatment had no effect on blood
pressure and cardiac hypertrophy but normalized the
arteriolar density. This indicates that AT2
receptors are involved in the angiogenesis response of the heart to Ang
II as previously reported in the chorioallantoic
membrane.9
Ang II–induced hypertension also affects capillary density in the
heart and confirms and extends previous studies showing that cardiac
hypertrophy secondary to pressure overload is associated
with a decrease in the capillary density in the left
ventricular
myocardium.3 4 19 Our data strongly
suggest that AT2 receptors are not involved in
the process, whereas pressor mechanisms and/or activation of
AT1 receptors by increased systemic Ang II
stimulate cardiomyocyte hypertrophy, which in
turn leads to a decrease in capillary supply.
The functional consequences of both the respective increase and
decrease in arteriolar and capillary densities as a result of Ang II
stimulation might be of importance in the long-term evolution of
hypertension-induced cardiac hypertrophy toward cardiac
failure. Whereas the capillaries do not contribute substantially to
resistance, the reduction in capillary density could reduce the oxygen
supply to cardiomyocytes and attenuate the coronary
flow reserve during prolonged enhanced ventricular work. On
the other hand, the increase in the number of arterioles in rats with
angiotensin-induced hypertension would lead to a decreased
coronary resistance and could therefore help to maintain the
balance between oxygen supply and consumption in hypertrophied
myocardium.
ACE
=
angiotensin-converting enzyme
AT1, AT2
=
angiotensin subtype 1 or 2 receptor
Ang II
=
angiotensin II
SM
=
smooth muscle
VSMC
=
vascular smooth muscle cell
vWf
=
von Willebrand factor
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