Bradykinin activity could explain the blood pressure increase during NaCl loading in hypertensive animals, but its contribution on vascular structure was not evaluated. We determined cardiac mass and large artery structure after a chronic, 4-mo, high-salt diet in combination with bradykinin B2-receptor blockade by Hoe-140. Four-week-old rats were divided into eight groups according to strain [spontaneously hypertensive rats (SHR) vs. Wistar-Kyoto (WKY) rats], diet (0.4 vs. 7% NaCl), and treatment (Hoe-140 vs. placebo). In WKY rats, a high-salt diet significantly increased intra-arterial blood pressure with minor changes in arterial structure independently of Hoe-140. In SHR, blood pressure remained stable but1) the high-salt diet was significantly associated with cardiovascular hypertrophy and increased arterial elastin and collagen, and2) Hoe-140 alone induced carotid hypertrophy. A high-salt diet plus Hoe-140 acted synergistically on carotid hypertrophy and elastin content in SHR, suggesting that the role of endogenous bradykinin on arterial structure was amplified in the presence of a high-salt diet.
- spontaneously hypertensive rats
studies on salt metabolism in hypertension have focused on the relationship between NaCl intake and the level of blood pressure, thus pointing to changes in the status of resistant arterioles. More recently, it has been recognized that excess salt is strongly associated with cardiac hypertrophy, a structural pattern observed in both hypertensive men and rats independently of the level of blood pressure (31). A temporal link between increased NaCl intake and aortic hypertrophy has been noted (17) in spontaneously hypertensive rats (SHR) in the absence of a significant change in blood pressure. However, the concomitant alterations in arterial smooth muscle mass and extracellular matrix have not yet been studied.
Structural changes of the vessels under high sodium intake may be related to sodium itself and/or to the subsequent hormonal changes associated with the diet, i.e., modifications of the renin-angiotensin and bradykinin activities. On the other hand, recent studies (13, 28) have shown that pharmacological and/or genetic suppression of several vasodilating systems, such as atrial peptides, nitric oxide, or bradykinin, could be responsible for increased salt sensitivity in animal models of hypertension. In particular, Majima et al. (20) showed that, in genetically bradykinin-deficient animals, blood pressure increased markedly when a high-salt diet was administered. Thus the role of endogenous bradykinin on NaCl-induced arterial changes has to be tested in such animal models of hypertension.
The purpose of the present study was, in Wistar-Kyoto (WKY) normotensive rats and SHR, to determine whether1) a chronic increase in salt intake is associated with equivalent structural changes of the carotid artery and the thoracic aorta in the two strains, and2) bradykinin B2 receptors might contribute to the observed structural modifications. For the latter purpose, the specific long-lasting inhibitor of bradykinin B2 receptors, Hoe-140 (18, 32), was used and assessed in a long-term study.
Forty-four male SHR and forty-eight WKY rats (Iffa-Credo, L’Abresle, France) were housed 5–7 per cage in our animal room (temperature, 20–22°C; humidity, 55–65%; 12:12-h light-dark cycles) and had free access to tap water. Each strain was randomly assigned into one of four groups (n = 10–12 per group) fed either low- or high-salt diets (0.4 or 7% NaCl) with or without B2-receptor blockade (Hoe-140 at a dose of 500 μg ⋅ kg−1 ⋅ day−1or placebo, respectively). Hoe-140 (Hoechst, Frankfurt, Germany) has already been described as a specific and selective inhibitor of bradykinin B2 receptors devoid of significant agonistic effect (18, 32). Both diets contained the same amount of potassium (0.73%). Placebo (isotonic saline) or Hoe-140 was injected subcutaneously twice daily. Salt diets and treatments were started simultaneously at the age of 4 wk and were continued for a period of 16 wk.
Arterial pressure and heart rate in conscious rats.
At the end of the 16-wk period, animals were anesthetized with pentobarbital (60 mg/kg ip). A catheter (PE-50 fused to PE-10; Guerbet Biomedical) was inserted in the lower abdominal aorta via the femoral artery, and the other end was filled with heparinized saline (50 U/ml) and tunneled under the skin of the back to exit between the scapulae. The animals were allowed to recover from anesthesia for 24 h in individual cages. The rats were hooked up to a monitoring device, and after at least a 30-min rest period, arterial pressure could be measured in conscious, freely moving rats in their own cages. Arterial pressure and heart rate were evaluated 12 h after the last drug administration, between 8:00 and 10:00 AM. Mean blood pressure (MBP) and heart rate were recorded by means of a Statham P23 ID pressure transducer (Gould, Cleveland, OH) connected to a Gould Brush recorder (G4133). The mean values of the continuous 2-h measurements were then calculated. At the end of these measurements, blood was drawn for the determination of plasma catecholamines (epinephrine, norepinephrine, dopamine) using a previously described radioenzymatic assay (26).
Histomorphometric study of thoracic aorta and carotid artery.
After blood samples were taken, the animals were anesthetized with pentobarbital, a median thoracotomy was performed, and animals were exsanguinated by means of a catheter placed in the right atrium while saline was injected through the femoral catheter. When the liquid exiting the auricle was clear, the circulatory system was perfused with a 4% formaldehyde solution. The animals died within seconds following the onset of formaldehyde infusion. After 1 or 2 min, a clamp was positioned on the auricle and the fixation liquid was allowed to infuse for 3 h at a pressure equal to the MBP of each animal (1, 15). The thoracic aorta and the carotid artery were then dissected out and stored in a 4% formaldehyde solution until the histological study was performed. The heart was also dissected and excised to determine left ventricular weight and then preserved in 4% formaldehyde.
The different structures of the vessels were examined in an arterial segment embedded longitudinally in paraffin. Three serial sagittal sections, 5 μm thick, were specifically stained to obtain a monochromatic color associated with the various structures of interest in the aortic media: Sirius red for collagen, orcein for elastin, and hematoxylin after periodic acid oxidation for the nucleus. Morphometric analysis was performed with a specialized automated image processor (NS 1500, Nachet-Vision, Paris, France), which is based on morphological mathematical principles and is software controlled. As previously described (1), different algorithms were developed to analyze each of the three structures (medial thickness; elastin; collagen) revealed by the specific staining in each of the three serial sections. For image processing, the image was transmitted to the processor via a video camera and could be viewed on the television monitor. The control of luminosity was automatically adjusted by the software to obtain similar contrasts, taking into account the total luminosity transmitted by the video camera. This analog image was then digitized as follows. Each elementary point (pixel) was automatically compared with a threshold. If the gray level of pixel exceeded this threshold, the pixel was given a numerical value of 1; otherwise it was given a numerical value of 0. Threshold parameters were defined by the size of such pixel groups and by their local contrast (top-hat transformation). The threshold was determined with the use of the top-hat transformation algorithm to minimize variations in nuclear staining and background.
Medial and luminal cross-sectional areas (CSA) were measured in arterial samples placed in a gel used for cryosections (medium inclusion ISOSYSTEM) and cooled to −20°C. When the gel solidified, some transverse sections of the arterial rings were cut from each sample. The sections were examined with a microscope and photographed at a known magnification (×48). When the films were developed, the pictures obtained were projected on a digitizer to allow measurement of the area of the vascular lumen and the external area of the vessel. The final magnification used (microscope amplification × projector magnification) was ×160. For each sample, the CSA of the vascular media and lumen were recorded. This latter parameter reflects the degree of arterial hypertrophy better than medial thickness does. Different studies have shown that the CSA of the arterial media is the most reliable constant of the vessel wall because it is not influenced by variations of the perfusion pressure (1).
Results are expressed as means ± SE. Data were analyzed using two-way analysis of variance (NaCl effect; Hoe-140 effect). To dissociate the effect of Hoe-140 from both the strain and the sodium effects, a three-way analysis of variance was performed on the histomorphometric parameters. For F< 0.05, Fisher’s exact test was applied for intergroup comparisons. After Bonferroni correction, a P value < 0.005 was considered to be significant.
Among the SHR, 12 rats died throughout the 4-mo treatment period. All these rats were on the high-salt diet (7 in the absence and 5 in the presence of Hoe-140). All these animals died between the sixth and tenth weeks of treatment in parallel with the development of congestive heart failure and/or stroke.
Blood pressure, left ventricular weight, and catecholamine levels.
Table 1 shows the changes in body weight, left ventricular weight, and hemodynamic variables. The high-salt diet was associated with a significant decrease in body weight in both rat strains but with an increase in the left ventricle-to-body weight ratio only in SHR (P < 0.005). After 4 mo of the high-salt diet, intra-arterial MBP was significantly increased in WKY rats but not in SHR (Fig. 1). None of these parameters were modified by Hoe-140 treatment.
As expected, plasma catecholamine levels are significantly higher in SHR than in WKY rats (P < 0.005). In SHR, dopamine, epinephrine, and norepinephrine levels were not modified by the high-salt diet and/or B2-receptor blockade (Table2). In WKY rats, norepinephrine values increased slightly with the high-salt diet (491 ± 129 vs. 298 ± 42 pg/ml; P = 0.04), but they were not affected by Hoe-140.
Arterial structure in WKY rats.
As shown in Table 3, there were minor changes in the histomorphometry of the carotid artery and the aorta in WKY rats. Thus, with the high-salt diet, the only change was an increase in carotid lumen. In combination with the high-salt diet, Hoe-140 was associated with a significant increase in the number of nuclei in the carotid artery.
Arterial structure in SHR.
Major histomorphometric changes in the carotid artery and the aorta were noted in SHR (Table 4). For both vessels, the high-salt diet was associated with significant increases in the arterial thickness and medial CSA. The aorta, but not the carotid artery, exhibited medial hypertrophy associated with cellular hypertrophy, as assessed by significantly larger nuclear area in high-salt fed rats. With the high-salt diet, extracellular matrix proteins (elastin and mainly collagen) were enhanced in both arteries. In response to Hoe-140, vessel thickness, medial CSA, and collagen and elastin contents of the carotid artery significantly increased. These latter changes were particularly observed in conjunction with the high-salt diet. More specifically, the effects of the high-salt diet on carotid artery medial hypertrophy and elastin content were accentuated by blockade of the bradykinin B2receptor. The three-way analysis of variance confirmed that, in SHR,1) Hoe-140 had a distinct effect on the content of the extracellular matrix, mainly elastin, and2) a synergistic effect with high-salt diet was observed for the carotid artery. Modifications related to medial thickness or nuclei were inconsistent or absent. This behavior was observed exclusively in SHR and not in WKY rats.
In the past, the effects of a high-salt diet on genetic hypertension were studied during short-term periods. Only the criterion of blood pressure was used as a gold standard to express salt sensitivity. In the present study, a long-term follow-up was performed in WKY rats and SHR, and the results, which were focused on cardiovascular structural changes, were mainly observed in SHR:1) a high-salt diet was associated with cardiovascular hypertrophy and increased elastin and collagen;2) blockade of bradykinin B2 receptors by Hoe-140 was associated with carotid hypertrophy, increased carotid elastin and collagen content, and accumulation of only collegen in the aorta; and3) in the carotid artery, the high-salt diet and Hoe-140 had a synergistic effect, enhancing arterial hypertrophy and extracellular matrix. In WKY rats, the high-salt diet significantly increased blood pressure but induced minor effects on carotid and aortic structure independently of the presence or absence of bradykinin B2-receptor blockade.
Hypertension is associated with hypertrophy of the heart and large conduit arteries and accumulation of extracellular matrix. Mechanisms that may contribute to arterial hypertrophy include hypertension per se, genetic factors, neural influences, and humoral factors (4, 11, 16,24). The finding that lowering blood pressure reverses arterial hypertrophy in aortic, carotid, or cerebral arteries suggests that an increase in pressure plays an important role in the development of vascular hypertrophy during chronic hypertension (24). However, hypertrophy and changes in the extracellular matrix of conduit arteries can occur independently of an increase in mean arterial pressure (1,9). Thus pressor and nonpressor effects are difficult to differentiate in animal models of hypertension.
In the present study, it is clear that hypertrophy of the heart, the aorta, and the carotid artery was present in SHR fed a high-salt diet and that intra-arterial blood pressure measured in conscious animals was not modified after 4 mo. Similarly, in stroke-prone (SP) hypertensive rats, significant structural changes of the cerebral arteries were observed with the high-salt diet and could be contained with a low-salt diet without any change in blood pressure (29). However, it is important to recognize that the lack of change in blood pressure in hypertensive rats does not mean that there was no contribution of mechanical factors to the observed structural changes. In this study, death occurred in 12 SHR fed the high-salt diet, suggesting that the lack of change in intra-arterial blood pressure was restricted to surviving animals. Thus the present data cannot be generalized but apply only to those SHR able to survive exposure to a high-salt diet. Whether the results are related to the resistance of these animals to increased blood pressure is not known. However, some reports in the literature (5) have shown an increase in blood pressure during follow-up of shorter duration in SHR. Mostly, high blood pressure is not the exclusive mechanical factor to consider in SHR fed a sodium diet. In this investigation, vascular hypertrophy in SHR was associated with either an unchanged (aorta) or increased (carotid artery) luminal CSA, suggesting different patterns of change in wall stress during the follow-up. However, in the presence of a high-salt diet, sodium overload is expected to be present, leading to changes in shear stress and geometric modifications of the vessels (4,8) independent of tensile modifications. Despite the fact that such difficulties were present, important differences between WKY rats and SHR may be assessed in this study. With a high-salt diet, WKY rats did not develop vascular hypertrophy despite the significant increase in the mean value of blood pressure, whereas SHR showed a significant degree of vascular hypertrophy in the absence of a substantial change in blood pressure (3-way analysis of variance).
Regarding the possible modifications of blood pressure under Hoe-140, it has been reported (23) that long-term Hoe-140 administration might decrease blood pressure when given alone to SHR. However, in that study, blood pressure was measured noninvasively on the tail artery. Because in rats and humans the mean value of arterial pressure remains constant along the arterial tree but pulse pressure increases markedly from central to peripheral arteries (30), measurements at the site of the tail artery do not reflect exactly what occurs in the central arteries. Changes in tail systolic blood pressure may be the simple consequence of an alteration of pressure wave transmission without any change in the mean arterial pressure of the thoracic aorta (30). In the present study, mean arterial pressure was measured intra-arterially in conscious animals, and the findings can exclude a long-term pressor effect of Hoe-140.
Interpretation of data.
In SHR, a temporal relationship between blood pressure elevation and the appearance of aortic thickening during salt loading was previously noted (17) as early as 5 wk of age. Blood pressure was not affected by the addition of salt for at least 11 wk, but vascular changes were significantly aggravated within 5 wk. Salt loading resulted in significant thickening of the aortic media between 10 and 20 wk of age. The present results agree with this finding but also showed, for the first time, that increased thickening was associated with a highly significant accumulation of elastin and collagen during the study period. Because some of the Hoe-140-induced structural changes might seem marginal in comparison with the sodium-induced and the hypertension-induced structural changes, a three-way analysis of variance was performed, enabling us to show that1) the Hoe-140-induced structural changes were independent of the effect of sodium intake and were observed exclusively in SHR, 2) a synergistic effect was observed with the high-salt diet at the site of the carotid artery of SHR, and 3) the nuclei and thickness modifications were inconsistent or absent.
In the literature, several findings indicate strong interactions between a high-salt diet and extracellular matrix accumulation in conduit arteries of rats with genetic hypertension. First, in Dahl salt-sensitive rats, carotid compliance was reduced in association with structural changes of the arterial wall but independently of transmural pressure levels (3). This reduction of compliance may be limited by diuretic treatment independently of changes in systemic blood pressure. Second, in SP hypertensive rats, enhanced carotid thickness and collagen accumulation were observed with a high-salt diet (29). Structural changes were contained by lowering NaCl intake while systemic blood pressure remained unchanged and survival was prolonged (7, 29). In SHR-SP rats, because chronic treatment with indapamide or hydrochlorothiazide does not induce any change of systemic blood pressure but is able to control the overexpression of some proteins such as smooth muscle α-actin, nonmuscle myosin, and type EIIIA fibronectin, salt and water depletion alone appears capable of reversing the typical phenotypic transformation from a contractile to a more synthetic pattern seen in untreated animals (7). Finally, in several models of genetic hypertension and depending on the importance of genetic salt sensitivity in each model, a high-NaCl diet is associated with the development of the secretory properties of smooth muscle cells, a situation in which the sensitivity to vasoactive compounds, such as bradykinin, is known to be potentially enhanced.
Although the role of endogenous bradykinin on cardiovascular structure in various experimental models has been previously published (10, 18,21), to the best of our knowledge, no report on such effects on large conduit arteries in SHR has been made. Recent studies (14, 21, 22, 25,27) have shown that kallikrein-like enzyme is present in cardiovascular tissue and that the kallikrein-kinin system acts as an autocrine mechanism in blood vessels. Madeddu et al. (19) showed that long-term blockade of bradykinin B2receptors by Hoe-140, when started early in life, altered the adult cardiovascular phenotype in rats. In the present study, the main results were observed in the carotid arteries of SHR, for which a strong interaction was observed between the high-salt diet and bradykinin B2-receptor blockade, leading to further arterial thickening and mostly enhancement of the extracellular matrix.
The mechanism(s) whereby, in the presence of a high-salt diet, bradykinin blockade has a synergistic effect on extracellular matrix is difficult to explain. Because bradykinin release interferes with the autonomic nervous system, it might be postulated that tropic influences of neural origin might act on the arterial wall. However, this possibility does not fit with the total lack of change of plasma catecholamines in SHR during the study period. A more plausible explanation might be that, with a high-salt diet, the resultant changes in salt overload and blood flow cause alterations in shear stress, endothelial function, and bradykinin release. Pharmacological studies (2, 6, 12, 21) have shown that, in the carotid artery and the thoracic aorta of normotensive and hypertensive rats, bradykinin (through B2-receptor stimulation and endothelial changes) was able to interfere with nitric oxide and to produce prostaglandin I2 and guanosine 3′,5′-cyclic monophosphate release. We hypothesize that these interactions could be responsible for the cardiovascular antihypertrophic effects of bradykinin and could even explain the differences between responses observed in the carotid and aortic walls.
In conclusion, the results of the present study demonstrate that the chronic high-salt diet in SHR was associated not only with cardiac but also with arterial hypertrophy, a phenomenon that occurred in the presence of minimal blood pressure changes. In combination with the high-salt diet, arterial hypertrophy was primarily attributable to enhanced extracellular matrix (elastin and mainly collagen) accumulation. On the basis of the effects of the specific antagonist Hoe-140, stimulation of bradykinin B2 receptors was associated with antihypertrophic effects and contributed to limitation of the extracellular matrix. In the carotid artery, the bradykinin-induced changes in arterial hypertrophy and elastin accumulation were amplified in the presence of the high-salt diet. This synergistic effect was not observed in the aorta, a finding which requires further investigation.
We thank Anne Safar for preparation of the manuscript. Hoe-140 was a gift from Hoechst Pharmacological (Frankfurt, Germany).
Address for reprint requests: M. Safar, Hôpital Broussais, Service Médecine 1, 96 rue Didot, 75674 Paris Cedex 14, France.
This study was supported by Institut National de la Santé et de la Recherche Médicale (Paris, France) and a grant from the Biomed Program of the European Community.
- Copyright © 1998 the American Physiological Society