(Hypertension. 1999;34:795-801.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Cardiac Microvasculature in DOCA-Salt Hypertensive Rats
Effect of Endothelin ETA Receptor Antagonism
From the MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montréal, Montréal, Quebec, Canada.
Correspondence to Ernesto L. Schiffrin, MD, PhD, FRCPC, Clinical Research Institute of Montreal, 110 Avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7. E-mail schiffe{at}ircm.qc.ca
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Abstract |
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Abstract—The cardiac abnormalities associated with hypertension include left ventricular hypertrophy and vascular changes. The latter may affect the cardiac microvasculature and predispose to myocardial ischemia. To test the hypothesis that endothelin-1 contributes to changes in the microcirculation of the heart, we studied cardiac microvessels of the deoxycorticosterone acetate–salt (DOCA-salt) model of hypertension in the rat, in which the endothelin system is activated, and the effect of the endothelin-A (ETA) subtype–selective endothelin receptor antagonist A-127722. A-127722 (30 mg · kg-1 · d-1) was administered for 4 weeks. Arterioles (
20 µm in lumen diameter) were identified in
the myocardium by use of immunolabeling with an
anti–smooth muscle 
-actin antibody, and capillaries with an
anti-laminin antibody with nuclear counterstaining by nuclear fast red.
Systolic blood pressure was 103±1.6 mm Hg in
unilaterally nephrectomized rats (UniNx), 202±3.2 mm Hg in
DOCA-salt (P<0.01 versus UniNx), and 182±3.1
mm Hg in ETA antagonist–treated DOCA-salt
(P<0.01 versus DOCA-salt or UniNx). Arteriolar and
capillary densities were altered significantly in the subendocardial
myocardium but not in the subepicardial
myocardium of the left ventricle. Arteriolar density per
square millimeter was 18.1±1.48 in UniNx, 31.9±3.26 in DOCA-salt
(P<0.01 versus UniNx), and 24.2±1.36 in
ETA antagonist–treated DOCA-salt (P<0.05
versus DOCA-salt or UniNx). Capillary density per square millimeter was
2395±148 in UniNx, 1576±107 in DOCA-salt (P<0.01
versus UniNx), and 1982±31 in ETA antagonist–treated
DOCA-salt (P<0.01 versus DOCA-salt or UniNx). In
conclusion, in DOCA-salt hypertensive rats, subendocardial arteriolar
growth and capillary rarefaction were observed in the left
ventricular myocardium, and both were partially
corrected by ETA receptor antagonism. This suggests a role
for endothelin-1 in cardiac arteriolar growth and capillary
rarefaction, which may have pathophysiological
implications by contributing to myocardial ischemia in
hypertension.
Key Words: microcirculation • arterioles • growth • capillaries • hypertension, experimental • myocardium • ischemia
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Introduction |
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Endothelin-1 (ET-1), a potent 21-amino-acid vasoconstrictor peptide produced by the endothelium of blood vessels, has been implicated as a mediator of blood pressure elevation1 and in the induction of vascular smooth muscle growth.2 3 These effects of ET-1 are mediated by the endothelin-A (ETA) subtype of endothelin receptor on vascular smooth muscle cells. The potential involvement of ET-1 in some models of experimental hypertension, such as deoxycorticosterone acetate (DOCA)–salt hypertension, has been suggested by the vascular overexpression of ET-1.4 5 Overexpression of ET-1 occurs in DOCA-salt hypertensive rats in blood vessels of different organs, including the heart, in which enhanced expression was found in the endothelium of epicardial and intramyocardial blood vessels and in the endocardium.6
Hypertension is often associated with cardiac complications. The major cardiac alteration is left ventricular hypertrophy, which may be implicated in the development of coronary insufficiency and arrhythmia and may lead to congestive heart failure.7 In addition, abnormalities that occur in coronary vessels are aggravated by hypertension. In humans, atherosclerosis at the level of epicardial coronary arteries is accelerated by hypertension and is often complicated by partial or complete occlusion of the vessel lumen and/or plaque rupture with or without thrombosis, leading to myocardial infarction. In addition, microvascular disease typically occurs in the heart in hypertension and may cause angina even in the presence of normal epicardial coronary arteries.8
The intramyocardial precapillary coronary vascular tree comprises small coronary arteries and arterioles. Small arteries present significant alterations in hypertension, as demonstrated in different hypertensive models: decreased lumen, increased media-to-lumen ratio, and sometimes increased media cross section.9 10 We previously showed that small intramyocardial arteries of DOCA-salt hypertensive rats present significant growth, that is, hypertrophic remodeling, with decreased lumen, increased media-to-lumen ratio, and increased media cross section. ET-1 is involved pathogenically in these changes, as shown by their reversal by treatment with endothelin antagonists.3 10 Coronary arterioles (lumen diameter <20 µm) play an important role in resistance to blood flow, and capillaries (lumen diameter <8 µm) are involved in oxygen/nutrient delivery to the myocardium.11 12 13 14 15 Abnormalities of the distribution, density, length, and tortuosity of these small vessels in hypertension may affect coronary vascular resistance and contribute to myocardial ischemia.16 17 18 19
The major aim of this study was to investigate (1) the effects of DOCA-salt hypertension on the density of coronary arterioles and capillaries in rat and (2) the effect of the ETA-selective endothelin receptor antagonist A-127722 on coronary arterioles and capillaries in this model, in which ET-1 has been shown to be involved.1 Specifically, we tested the hypothesis that ET-1 plays a role in myocardial microvascular abnormalities in DOCA-salt hypertensive rats via activation of ETA receptors.
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Methods |
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Animal Experiments
The study was approved by the Animal Care Committee of the Clinical Research Institute of Montreal and conducted in accordance with the recommendations of the Canadian Council of Animal Care. DOCA-salt hypertension was induced by the method of Ormsbee and Ryan.20 Briefly, male Sprague-Dawley rats (Charles River, St Constant, Québec, Canada) weighing 200 g were unilaterally nephrectomized under sodium pentobarbital anesthesia (40 mg/kg). Silicone rubber impregnated with DOCA (Sigma Chemical Co) (200 mg/rat) was implanted subcutaneously, and rats were offered 1% saline to drink. Control rats (UniNx) were also uninephrectomized but received a silicone rubber implant without DOCA and tap water to drink. The ETA endothelin receptor antagonist A-127722 was administered to DOCA-salt rats in the drinking water (30 mg · kg-1 · d-1) for 4 weeks. After rats were warmed, systolic blood pressure was measured weekly by the tail-cuff method under slight restraint. Readings were recorded on a model 7 polygraph fitted with a 7-P8 preamplifier and PCPB photoelectric pulse sensor (Grass Instruments Co). The average of 3 pressure readings was obtained. Rats were killed by decapitation at the end of the experiment, and the hearts were fixed in Bouin's fixative.
Immunohistochemistry
To study arterioles and capillaries in the
myocardium, the histochemical approach to identify these,
described by Sabri et al,21 was modified as follows. The
antibodies used were a mouse monoclonal antibody against smooth muscle
-actin (Sigma) and rabbit polyclonal antibody directed against
laminin (Sigma). The hearts, fixed in Bouin's fixative, were
processed for paraffin embedding in an automated system (Shandon
Citadel tissue processor). Serial sections (5 µm) of the median
part of the left ventricle were obtained. The tissue sections were
dewaxed with ethanol and then blocked with 10% normal goat serum or
10% normal sheep serum for anti-laminin and anti–smooth muscle
-actin antibodies, respectively, for 1 hour at room temperature to
reduce nonspecific binding. Three sections of the left ventricle from
each rat were incubated with anti-laminin IgG (1:500) or anti–smooth
muscle -actin IgG (1:300) for 16 hours at 4°C. The sections were
washed thoroughly 3 times with PBS and incubated with secondary
biotinylated antibodies (1:100) for 1 hour at room temperature, with
goat anti-rabbit IgG (Vectastain ABC Kit, Vector Laboratories) for
laminin, and with sheep anti-mouse IgG (Amersham) for smooth muscle
-actin. The tissue sections were rinsed with PBS and incubated with
streptavidin–horseradish peroxidase complex (Vectastain ABC Kit,
Vector Laboratories) at 1:100 for 1 hour at room temperature. After 3
washes with PBS, the sections were developed with 3,3'-diaminobenzidine
in Tris-HCl-H2O2. Sections
labeled with anti-laminin IgG were also counterstained with nuclear
fast red (ICN) for 5 minutes.
Determination of Arteriolar and Capillary Density
In every heart, microscopic analysis of arteriolar and
capillary density with a video imaging program (Northern Eclipse 5.0,
EMPIX Imaging Inc) was performed on 3 nonconsecutive serial sections
(which allowed convergence of results) of the median part of the
ventricle. Vessel density was evaluated throughout the inner third
(subendocardial myocardium) and the outer third
(subepicardial myocardium) of the circumference of the left
ventricle. From each section, 15 to 35 fields (magnification x100 and
x20 for capillaries and arterioles, respectively) were recorded.
Quantitative analyses were performed by a blinded observer.
Capillary density was determined in sections labeled with laminin
antibody counterstained with nuclear fast red and was based on the
quantification of positively labeled structures with <8-µm lumen
size and with 1 nucleus (endothelial cell). Arterioles
were quantified in sections that were immunolabeled with smooth muscle
-actin antibody. Only small blood vessels with a lumen diameter of
<20 µm surrounded by 1 smooth muscle layer that stained
positively were counted.
Data Analysis
Data per heart were the means of the density of vessels from 3
sections. Data are expressed as mean±SEM. Statistical significance was
assessed by 1-way ANOVA followed by a Student-Newman-Keuls test or in
the case of proportions by 
2 test. A value of
P<0.05 was considered significant.
To evaluate the tortuosity of arterioles, the mathematical model
proposed by Adair et al22 was used. The relationship
between sampling probability and sine of the orientation angle is the
basis of the method: 1/sin is used as a weighting factor (W) to
correct for the decreased sampling incidence caused by orientation of
vessels. Vessels are modeled as cylindrical tubes, and their profile is
treated as the elliptical intersection of the vessel with the section
plane. As a result, W=a/b, where a is the major axis of the ellipse and
b is the minor axis of the ellipse. Only vessels with a/b<2.5 were
used for the calculation, to eliminate the arterioles sectioned along
their longitudinal axis.21 22 Therefore, Lv (length
density) can be calculated as Lv=(
a/b)/At, where At is the
test area. A series of individual sections are treated as 1 large
representative section, and test areas and individual W
values are summed. Tortuosity was assessed quantitatively by use of an
anisotropy coefficient calculated as Lv/Na, where Na is numerical
density (number of profiles per cross-sectional area). An anisotropy
coefficient of 1.0 indicates a total absence of tortuosity, ie, all
vessels are parallel to each other, whereas an anisotropy coefficient
of 2.0 indicates maximum tortuosity, ie, the orientation of the vessels
is entirely random.
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Results |
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Blood Pressure and Body and Heart Weights of Rats
The systolic blood pressure of DOCA-salt hypertensive rats was significantly elevated relative to that of UniNx control rats after 4 weeks (Table 1). Treatment with the endothelin antagonist A-127722 resulted in slightly but significantly lower blood pressure in DOCA-salt hypertensive rats compared with untreated DOCA-salt rats. The body weight of DOCA-salt hypertensive rats was significantly lower than that of normotensive control rats. Relative heart weights were similar in treated and untreated DOCA-salt hypertensive rats and higher than those of normotensive rats, indicating that the endothelin antagonist had no effect on cardiac hypertrophy.
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Characterization of the Microvasculature in Subendocardial and
Subepicardial Myocardium of the Rat Left Ventricle
Figure 1 shows the 2 types of small
vessels that were characterized by immunohistochemical techniques.
Laminin staining delineated cardiomyocytes and blood
vessels, and subsequent nuclear fast red counterstaining revealed the
endothelial cell layer in capillaries, as indicated by
the presence of only 1 nucleus per vessel. Smooth muscle -actin
labeling distinguished arterioles from capillaries, because the latter
do not express smooth muscle -actin.
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Arteriolar and Capillary Density in DOCA-Salt Subendocardial and
Subepicardial Myocardium: Effect of A-127722
Arteriolar and capillary densities were not significantly
different in the subepicardial myocardium of the left
ventricle of DOCA-salt hypertensive rats compared with hearts from
UniNx controls (Figure 2, top). In
contrast, in the subendocardial myocardium, arteriolar
density was higher and capillary density was lower in DOCA-salt
compared with that in hearts of UniNx control rats (Figure 2, bottom). A-127722 partially attenuated the changes in subendocardial
density of capillaries and arterioles in the DOCA-salt rat. To
determine whether changes in arteriolar density could be attributable
to a shift in the diameter of arterioles to a greater number of vessels
<20 µm in DOCA-salt hearts, which would have artifactually
increased the density of vessels counted, the frequency distribution of
diameters of arterioles <30 µm was evaluated (Figure 3). There were some significant minor
differences in distribution (more arterioles of <10 µm in
DOCA-salt rat hearts, fewer of 15 µm), but a
2 test showed that proportions of vessels <20
or >20 µm were similar in the 3 groups for lumen diameters of
either 15 to 20 µm versus 20 to 25 µm or 10 to 20
µm versus 20 to 30 µm. The increase in arteriolar density in
DOCA-salt rat hearts could therefore not be attributed to increased
numbers of arterioles <20 µm. Thus, we indeed detected
arteriolar growth and capillary rarefaction in the left
ventricular subendocardial myocardium of the
DOCA-salt hypertensive rat, and both were partially corrected by
administration of the ETA receptor
antagonist A-127722.
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Analysis of the tortuosity of arterioles in the subendocardial region of the myocardium, where the density of arterioles was significantly increased, did not show any significant differences in the anisotropy coefficient between groups (Table 2).
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Discussion |
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The present study investigated the microvasculature of the subendocardial and subepicardial myocardium of DOCA-salt hypertensive rats and the potential role that endothelin may play in the changes found in relation to normotensive Uni-NX control rats. DOCA-salt hypertensive rats exhibited increased arteriolar density and decreased capillary density in the subendocardial layer of the myocardium but not in the subepicardial myocardium, and ETA-receptor antagonism partially corrected these microvascular abnormalities. As demonstrated by data in Figure 3, arteriolar density changes could not be attributed to a shift in diameter of arterioles to a greater number of vessels <20 µm in DOCA-salt hearts, which would have resulted in a greater number of arterioles counted, because distribution of arteriolar sizes <20 or >20 µm was similar in the 3 groups for lumen diameters of 15 to 20 versus 20 to 25 µm or 10 to 20 versus 20 to 30 µm.
The site of origin of new arterioles within the microvascular network remains unknown. Although it is widely accepted that capillaries grow by sprouting into the interstitium,23 24 an angiogenic sprouting process has not been observed in arterioles. The current hypothesis for arteriolar development is that certain capillaries, selected by presently unknown mechanisms, become invaded with vascular smooth muscle cells and are therefore transformed into arterioles, a process called arterialization.25 Arterialization may occur by several possible mechanisms. Nelhs et al24 have suggested that pericytes surrounding capillaries may be dormant smooth muscle precursors. These cells may differentiate to smooth muscle cells, resulting in the formation of "meta-arterioles." Alternatively, vascular smooth muscle cells in preexisting arterioles may migrate downstream to arterialize capillaries. The precise stimuli that initiate the process of arterialization are as yet unclear. Hypoxia, hemodynamic stresses, and hormonal stimuli may participate to different degrees in the stimulation of arteriolar development. Analysis of the tortuosity of arterioles in the subendocardial region of the myocardium, where the density of arterioles was significantly increased, using the mathematical approach proposed by Adair et al22 did not show any significant differences in the anisotropy coefficient between groups. Thus, increases in subendocardial arteriolar density, although probably associated with lengthening of arterioles as a result of arterialization of capillaries, did not result in more tortuous vessels, probably because of the cardiac hypertrophy occurring in DOCA-salt hypertensive rats.
ET-1 elicits a variety of biological effects that include vascular smooth muscle cell contraction26 and growth.2 3 ET-1 has also been shown to induce smooth muscle cell migration in the presence of low concentrations of platelet-derived growth factor or angiotensin II.27 The potentiation of vascular smooth muscle cell migration by ET peptides was inhibited by the ETA receptor antagonist BQ-123 in a concentration-dependent manner. These results suggest that the endothelin family of peptides, especially ET-1, can induce human coronary artery smooth muscle cell migration in combination with platelet-derived growth factor or angiotensin II, probably via the ETA receptor.26 The partial correction of abnormal arteriolar density by ETA receptor antagonism in the present study implicates ETA receptors as mediators of effects of ET-1 in the arterialization process that occurs in the hearts of DOCA-salt hypertensive rats.
Decreased capillary density, or rarefaction, has been described in many vascular beds and different models of hypertension16 17 and under the effects of in vivo angiotensin II infusion.21 Rarefaction is considered to proceed through a stage of functional rarefaction (reversible) caused by arteriolar spasm and diversion of blood flow away from some capillaries. This may be followed by anatomic rarefaction, with definitive closure of capillaries. One can envisage that the potent vasoconstrictor effect of ET-1 may result in constriction and closure of meta-arterioles, resulting in diversion of blood flow and capillary collapse. This phenomenon may be compounded by the increased resistance to flow resulting from the lengthening of the high-resistance arteriolar tree as a result of the arterialization of capillaries. The end result will be anatomic rarefaction. Blockade of ETA receptors appears to successfully reverse this process, at least in part, suggesting that to some degree, the rarefaction may be functional rather than anatomic at the stage at which intervention with the ETA antagonist was initiated. A potential model for the increased density of arterioles and decreased density of capillaries in the subendocardial myocardium is depicted in Figure 4.
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A decrease in capillary density may compromise oxygen and nutrient supply to cardiac myocytes by shortening and reduction of the numbers of capillaries and therefore time capacity of diffusion. Vulnerability to hypoxia is greater in the subendocardial myocardium of the left ventricle than in the subepicardial myocardium.28 29 Interestingly, microvascular abnormalities were localized specifically to the subendocardial region, the more vulnerable area of the myocardium. Moreover, the increase in arteriolar density could lead to an increase in coronary vascular resistance through an increase in the length of the arteriolar segment of the vascular tree (see model in Figure 4), thus contributing to hypoxic events. The localization of the growth (arterioles) and rarefaction process (capillaries) to the subendocardial, more vulnerable, region may suggest that the process is multifactorial. Hemodynamic (including pressure) and metabolic variables probably contribute, together with ET-1 and presumably other hormonal stimuli, to the vascular growth and rarefaction process found in this region of the heart of DOCA-salt hypertensive rats.
There is some evidence that ET-1 may induce growth of the heart and contribute to left ventricular hypertrophy30 and collagen deposition.31 Cardiac hypertrophy could therefore have a confounding effect on the arteriolar and capillary changes found after endothelin antagonist treatment in DOCA-salt hypertensive rats, because this hypertensive model presents rather severe left ventricular hypertrophy. In this and previous studies, we found that although ET-1 is overexpressed in the heart of DOCA-salt hypertensive rats,6 treatment with either balanced ETA/ETB3 or selective ETA endothelin receptor antagonists10 was not associated with improvement of cardiac hypertrophy. This may be explained in part by the fact that in the DOCA-salt hypertensive rat, the increase in ET-1 gene expression occurs in the endothelium of coronary arteries and endocardium rather than the myocardium or interstitium.6 Moreover, blood pressure is lowered only moderately in the endothelin antagonist–treated rats, and more effective blood pressure lowering may be necessary for endothelin antagonism to have a clinically evident effect on cardiac hypertrophy. Consequently, in the absence of differences in cardiac hypertrophy between treated and untreated DOCA-salt hypertensive rats, the microvascular changes found, characterized by increased density of arterioles and decreased density of capillaries in the subendocardial region of the left ventricle, cannot be attributed to cardiac hypertrophy. A limitation of the study is that differences in cellular composition and collagen deposition were not examined and could potentially contribute to the changes found. Remodeling of the heart; apoptosis, which is enhanced in this model, particularly after treatment with endothelin antagonists32 ; and fibrosis could result in alterations in vascular density. Although this cannot be excluded, it would be unlikely that increased arteriolar density and decreased capillary density limited to the subendocardium in the untreated DOCA-salt hypertensive rats would be corrected, in part as a result of a combination of these phenomena in the endothelin antagonist–treated rats. Systolic blood pressure rise was moderately reduced by 20 mm Hg in DOCA-salt hypertensive rats by treatment with the ETA antagonist. Although the possibility of a role of lower blood pressure affecting arteriolar growth and capillary rarefaction in the heart cannot be eliminated,13 16 17 21 it seems improbable, considering that cardiac hypertrophy was unaffected by the lower blood pressure.
In conclusion, in DOCA-salt hypertensive rats, subendocardial arteriolar growth and capillary rarefaction were observed in the left ventricular myocardium, and both were partially corrected by ETA receptor antagonism. This suggests a role for ET-1 in cardiac arteriolar growth and capillary rarefaction in the heart of DOCA-salt hypertensive rats, which may have pathophysiological implications in hypertension by contributing to myocardial ischemia.33 Together with previous data suggesting that ETA antagonism improves vascular reactivity in the coronary circulation,34 the present study suggests that blockade of the endothelin system at the level of ETA receptors exerts beneficial cardiac effects in forms of hypertension, such as the DOCA-salt rat, in which the endothelin system is activated.
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Acknowledgments |
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This study was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension. The authors are grateful to André Turgeon for his excellent technical assistance.
Received May 9, 1999; first decision June 15, 1999; accepted June 30, 1999.
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M. Iglarz, R. M. Touyz, F. Amiri, M.-F. Lavoie, Q. N. Diep, and E. L. Schiffrin Effect of Peroxisome Proliferator-Activated Receptor-{alpha} and -{gamma} Activators on Vascular Remodeling in Endothelin-Dependent Hypertension Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 45 - 51. [Abstract] [Full Text] [PDF]
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A. D. Giulumian, M. M. Molero, V. B. Reddy, J. S. Pollock, D. M. Pollock, and L. C. Fuchs Role of ET-1 receptor binding and [Ca2+]i in contraction of coronary arteries from DOCA-salt hypertensive rats Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1944 - H1949. [Abstract] [Full Text] [PDF]
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