Microvasculature in Angiotensin II–Dependent Cardiac Hypertrophy in the Rat
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.
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 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
The antibodies used were monoclonal antibodies directed against human SM α-actin (Dako) or SM α-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 α-actin or SM α-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 ×100) 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 α-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 α-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.
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⇓). Losartan but not PD123319 treatment prevented these Ang II–induced changes in arterial pressure and heart weight to body weight ratio.
Characterization of Microvasculature in Endomyocardium of Rat Left Ventricle
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 α-actin and SM α-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.
Effects of Ang II on Endomyocardial Microvasculature
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.
In the endomyocardium of the left ventricle, the number of arterioles positively stained with both SM α-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).
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 α-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.
Changes in Arteriolar Density
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 α-actin and SM α-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
Changes in Capillary Density
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.
Selected Abbreviations and Acronyms
|AT1, AT2||=||angiotensin subtype 1 or 2 receptor|
|Ang II||=||angiotensin II|
|VSMC||=||vascular smooth muscle cell|
|vWf||=||von Willebrand factor|
This work was supported by INSERM, CNRS, European Union (Biomed), and Fondation de France.
- Received January 24, 1998.
- Revision received February 10, 1998.
- Accepted April 17, 1998.
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