Abstract We previously showed that endothelin-1 expression was increased in vascular endothelium of deoxycorticosterone acetate–salt hypertensive rats, whereas in spontaneously hypertensive rats (SHR) it is similar to or less than that in normotensive rats. Treatment with the combined endothelin type A/endothelin type B receptor antagonist bosentan moderately reduced blood pressure rise and nearly completely blunted the development of vascular hypertrophy, particularly in small arteries, in the deoxycorticosterone acetate–salt hypertensive model, suggesting a paracrine role for vascular endothelin-1 in the induction of blood vessel hypertrophy in some forms of experimental hypertension. In the present study we examined the effect of chronic oral treatment for 4 weeks of 12-week-old SHR and Wistar-Kyoto rats (WKY) with 100 mg/kg per day bosentan. Blood pressure rose to hypertensive levels similarly in bosentan-treated and untreated SHR; systolic pressure of WKY was also unaffected. The wet weights of the heart, of aortic segments, and of the mesenteric arterial bed were similar in treated and untreated SHR. When coronary, renal arcuate, mesenteric, and femoral small arteries were evaluated on a wire myograph, the media width and media-to-lumen ratio were greater and the lumen diameter was smaller in vessels from SHR relative to those from WKY, except in small arteries from the renal cortex, in which the lumen was not significantly different in both strains. The media cross-sectional area of small arteries from the four vascular beds was similar in both strains. Identical morphometric parameters were found in the four vascular beds in bosentan-treated and untreated rats of each strain. We conclude that endothelins do not play a role in the maintenance of elevated blood pressure or in vascular hypertrophy or remodeling in adult SHR. This study also showed similar morphometric changes consistent with remodeling in small arteries of the coronary, mesenteric, and femoral vascular beds of SHR, whereas the changes in renal cortical small arteries were consistent with vascular hypertrophy.
Endothelins are potent vasoconstrictors1 endowed with hypertrophic and mitogenic potential.2 3 Of the endothelin family, endothelin-1 (ET-1) is produced by endothelial cells and may act on underlying smooth muscle cells, which bear endothelin type A (ETA) and endothelin type B (ETB) receptors.4 Both receptor subtypes may mediate the vasoconstrictor effects of endothelins.5 Endothelins also interact with endothelial ETB receptors, which induce the generation of endothelium-derived nitric oxide and prostacyclin.6 Vascular responses to ET-17 and endothelin receptors and signaling8 9 are reduced in deoxycorticosterone acetate (DOCA)–salt hypertensive rats, and we have speculated that the receptor downregulation found could result from enhanced vascular production of ET-1. ET-1 gene expression10 and the content of immunoreactive ET-111 have been shown to be increased in blood vessels of DOCA-salt hypertensive rats. Chronic treatment of DOCA-salt hypertensive rats with the new combined ETA/ETB receptor antagonist bosentan resulted in a reduced rise in blood pressure associated with attenuation of vascular hypertrophy, particularly at the level of mesenteric small arteries, where the normalization of structure exceeded what could be expected from the slightly lower blood pressure reached.12 This suggested a specific role of vascular ET-1 overexpression in the vascular hypertrophy in this model of experimental hypertension and prompted us to investigate the potential role of ET-1 on blood pressure elevation and in vascular changes in spontaneously hypertensive rats (SHR).
In SHR, ET-1 expression in blood vessels is similar to or less than that of age-matched Wistar-Kyoto rats (WKY).11 This agrees with the prior demonstration of normal or only slightly depressed responses to ET-1 and the absence of important downregulation of endothelin receptors in blood vessels or smooth muscle cells of SHR.13 14 15 16 To determine whether there was a relationship between the response of blood pressure and that of vascular hypertrophy and remodeling to treatment with the combined ETA/ETB receptor antagonist bosentan in SHR, we designed this study in which SHR and WKY were treated chronically with bosentan. Effects of treatment on blood pressure and hypertrophy of the heart, conduit blood vessels, and small arteries of the coronary, renal, mesenteric, and femoral vasculature were evaluated.
Animal experiments were performed following the recommendations of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the Clinical Research Institute. SHR and control WKY were bought from Taconic Farms (Germantown, NY) and were received at 10 weeks of age. Rats were housed under conditions of constant temperature (22°C) and humidity (60%) and exposed to a 12-hour light/dark cycle. Systolic pressure was measured weekly by the tail-cuff method, after rats were warmed and under slight restraint, and recorded on a model 7 polygraph (Grass Medical Instruments) fitted with a 7-P8 preamplifier, using a PCPB photoelectric pulse sensor. The average of three pressure readings was recorded. Rats were offered, starting at 12 weeks of age and for 4 weeks, the combined ETA/ETB receptor antagonist bosentan17 (kindly provided by Dr Martine Clozel, Hoffmann–La Roche, Basel, Switzerland) at a daily oral dose of 100 mg/kg body wt mixed with powdered chow. This oral dose blocks the action of pressor doses of intravenously injected big ET-1 for more than 24 hours17 and is the dose that reduced blood pressure and resulted in blunting of the development of vascular hypertrophy in DOCA-salt hypertensive rats.12 Additionally, a parallel group of four SHR and six WKY was either offered or not offered this daily dose of bosentan with their food for 4 to 5 days. Then, with these rats under pentobarbital anesthesia (50 mg/kg IP), saline-filled PE-50 catheters (Clay Adams) were introduced into the jugular vein and carotid artery. Bolus intravenous injections of 50 ng angiotensin II and of 300 pmol/kg ET-1 (both from Peninsula Laboratories) separated by at least 30 minutes were administered. Blood pressure was monitored with Statham P23 ID pressure transducers (Gould Inc) and recorded on a Grass model 7 polygraph.
Preparation of Small Arteries
On the day of the experiment, rats were killed by decapitation. The heart and kidneys were removed and placed in ice-cold Krebs’ solution. The rat was then placed in the supine position, and the skin of the right hind leg was incised. An artery in the popliteal region of approximately 2 mm in length was dissected. For dissection of coronary vessels, the right ventricle was opened to expose coronary arteries on the interventricular septum. The interventricular artery was followed to the cardiac apex, and then the chordae tendinae and myocardium were separated and a vessel 2 mm in length was isolated. For the isolation of renal cortical arteries, the renal capsule was first removed. The kidney was sectioned, and a renal artery was dissected close to the renal cortex and then followed distally. A renal arcuate artery approximately 2 mm in length was isolated. Mesenteric small arteries were obtained as previously described.7 12 14 Superior mesenteric arteries were taken from the part of the mesenteric vascular bed that feeds the jejunum 8 to 10 cm distal to the pylorus. A third-order branch at a distance of 1 mm from the intestine and approximately 2 mm in length was isolated. The vessels were mounted as ring preparations on an isometric myograph (Living Systems Instrumentation). Dissection and mounting were performed in physiological salt solution (PSS) at room temperature. The PSS had the following composition (mmol/L): NaCl 120, NaHCO3 25, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5, EDTA 0.026, and glucose 5.5. All solutions were bubbled with 95% O2 and 5% CO2 to give a pH of 7.40 to 7.45. Solutions were maintained at 37°C.
Protocol of Study of Small Arteries
After mounting, the vessels were warmed to 37°C and allowed to equilibrate in PSS for approximately 30 minutes with the vessel internal circumference set to give a wall tension of 0.2 mN/mm. Then media width was measured with a Leitz-Diavert inverted light microscope (Wild Leitz) at a magnification of ×320 at 12 different sites along the wall, which were then averaged. The vessels were then set to L0, where L0=0.9 · L100, and L100 is the internal circumference (calculated from the distance between the wires) that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. After this, the vessels were maintained in PSS at 37°C for a further 90 minutes. After the rest period, the vessels were stimulated with 10 μmol/L norepinephrine or 10 nmol/L arginine8 vasopressin (in the case of coronary small arteries) to ensure that the vessels isolated were arteries and developed a tension of more than 2 mN/mm.
Blood was obtained from the neck, in tubes containing potassium ethylenediaminotetraacetate, during the first few seconds after decapitation for measurement of plasma ET-1. Immunoreactive ET-1 was extracted from plasma by passage through C18 Sep-Pak cartridges (Waters Associates) and measured by radioimmunoassay as previously described.15 18
Analysis of Data
The media cross-sectional area of small arteries (A) was obtained from the media thickness (m) and the circumference of vessels (L), all measured with the vessel relaxed and under no passive stretch (wall tension of 0.2 mN/mm) and was calculated as A=Lm+πm2. Using L0 and the calculated media cross-sectional area, and assuming a constant media volume, the standardized media thickness of blood vessels (at L0) was then calculated. The lumen diameter was obtained as L0/π. In preliminary experiments, we established that for normotensive rats weighing 350 and 460 g, the only vascular parameter that differed significantly according to body weight was the cross-sectional area of the media. In normotensive rats weighing 460 g, the square root of the ratio between 350 and 460 g (0.87) provided an empirical factor that, when multiplied by the cross-sectional areas of the media of small arteries of these rats, rendered them equal to the cross-sectional areas of the mediae of small arteries of the normotensive rats weighing 350 g. The cross- sectional areas of the media of small arteries of WKY were therefore also multiplied by this value, and the results of this correction for differences in body weight of SHR and WKY are provided in parentheses where applicable.
Values are given as mean±SEM. Statistical differences were evaluated by ANOVA followed by a Newman-Keuls test. Results were considered significantly different at a value of P<.05.
Effectiveness of Endothelin Receptor Blockade by the Oral Dose of Bosentan Administered Chronically
SHR and WKY that had received or not received bosentan at the dose of 100 mg/kg per day mixed with their powdered chow for 4 to 5 days were investigated with rats under anesthesia. Intravenous bolus injections of 50 ng angiotensin II induced an identical rise in blood pressure in WKY and SHR, whether or not they were receiving bosentan (Fig 1⇓). ET-1 at a dose of 300 pmol produced an initial decrease in blood pressure, followed by an elevation of blood pressure in rats not receiving bosentan orally with their food. In bosentan-treated rats, the transient ET-1–induced vasodilation preceding the vasoconstriction was abolished in WKY and reduced in SHR by 40%. The pressor effect induced by ET-1 was reduced by 60% (Fig 1⇓), similar to what has been reported previously.17
Systolic Pressure, Weight, and Plasma Hormone Concentrations
Systolic pressure of treated and untreated SHR was significantly higher than that of age-matched WKY (Fig 2⇓). There was no difference in blood pressure between bosentan-treated and untreated rats within the same strain. Thus, bosentan had no effect on blood pressure of SHR or WKY. SHR had a lower body weight than age-matched WKY (Table 1⇓), as we have found in previous studies.14 15 Heart weight and the ratio of heart weight to body weight were greater in SHR than in WKY and were unaffected by bosentan treatment. Plasma immunoreactive ET-1 concentration was similar in SHR and WKY and exhibited a trend toward an increase, albeit without achieving statistical significance, in bosentan-treated WKY. No change in plasma immunoreactive ET-1 occurred in bosentan-treated SHR.
Wet Weight of Aorta and the Mesenteric Arterial Bed
The wet weights of a 1.5-cm-long segment of aorta and of the complete mesenteric arterial bed were slightly lower in SHR than in WKY, as we have reported previously15 (Table 2⇓). No difference was present between bosentan-treated and untreated rats of each strain.
Coronary, Renal, Femoral, and Mesenteric Resistance Artery Structure
The lumen diameter (Fig 3⇓) of small arteries dissected from the coronary (interventricular septum), femoral (popliteal), and mesenteric circulations was smaller in SHR than in WKY, but the difference did not achieve statistical significance in renal small arteries (arcuate arteries). The media width (Fig 4⇓) and media-to-lumen ratio of coronary, renal, femoral, and mesenteric small arteries (Table 3⇓) were greater in SHR than in WKY. The cross-sectional area of the media of small arteries of the four vascular beds was similar in SHR and WKY (Table 3⇓). When media cross-sectional area of the media of WKY was corrected for the greater body weight of these rats (values in parentheses in Table 3⇓), that of the renal small arteries of SHR was greater than that of WKY, whereas in other vascular beds, differences reached significance only at the .05 level in some of the groups (treated or untreated, but not in both). Bosentan treatment did not result in any significant differences in any of these morphometric parameters in either SHR or WKY (Figs 3⇓ and 4⇓, Table 3⇓).
The results of this study indicate that chronic oral treatment with the combined ETA/ETB receptor antagonist bosentan has no effect on blood pressure, cardiac hypertrophy, or conduit or resistance artery hypertrophy and remodeling in SHR. Together with previous results showing that in SHR ET-1 expression in blood vessels is normal or reduced,11 that plasma concentrations are not increased,11 14 and that responses to ET-1 of blood vessels13 14 15 or vascular smooth muscle cells16 are normal or slightly depressed, these data unambiguously indicate that ET-1 does not have a role in the maintenance of elevated blood pressure in this genetic model of hypertension. These data also demonstrate conclusively that in contrast to DOCA-salt hypertensive rats, in which ET-1 is involved in vascular hypertrophy, in SHR, ET-1 plays no discernible role in vascular hypertrophy or remodeling.
Studies with BQ-123, an ETA receptor antagonist, have demonstrated a slight lowering of blood pressure in SHR.19 The lack of effect of the combined ETA/ETB receptor antagonist bosentan in SHR that we report in the present study could be due to blockade of endothelial ETB receptors and reduction in the generation of endothelium-derived nitric oxide or prostacyclin, which are vasorelaxants. This could counteract the effect of the antagonist on ETA receptors. However, this does not appear to be the case, because in DOCA-salt hypertensive rats, blood pressure does decrease with bosentan treatment.12 Since the effects of BQ-123 were obtained by intravenous injection in acute experiments and the present results are from chronic oral administration of bosentan, this difference in the route of administration and time frame of the experiments may explain the discrepancy observed. That the bosentan dose provided to the rats is adequate to block responses to exogenous endothelins has already been demonstrated17 and is again shown by the present experiments (Fig 1⇑). Furthermore, in our previous study,12 performed similarly to the current one, bosentan was administered identically, at the same dose, and induced a blunting of the elevation of blood pressure and abrogated the development of vascular hypertrophy and remodeling in DOCA-salt hypertensive rats, showing the ability of this form of bosentan administration to antagonize the effects of enhanced endogenous production of ET-1 in that hypertensive model.10 11 The absence of increased endothelin content in blood vessels of SHR11 and the normal or reduced abundance of ET-1 mRNA in arteries of these hypertensive rats, which has been recently demonstrated,20 would allow us to predict that the negative result reported is the expected one. Together with evidence that responses of blood vessels to ET-1 are normal or blunted in SHR,14 15 16 these results also contribute to establishing the fact that the role played by ET-1 is an additional pathophysiological difference between SHR and the DOCA-salt hypertensive model. Thus, ET-1 does not appear to be involved in hypertension in this genetic model of high blood pressure, whereas in the DOCA-salt hypertensive rat, ET-1 may play a critical role in vascular hypertrophy and blood pressure elevation. However, it remains to be established whether long-term treatment starting before the development of hypertension in SHR may reveal some role for endothelins in this genetic model of hypertension.
Previous studies have demonstrated that after treatment with combined ETA/ETB receptor antagonists, the concentration of immunoreactive ET-1 in plasma increases significantly.12 21 The mechanism for this rise is unclear, but displacement of ET-1 bound to ETB receptors on the endothelium has been proposed as one potential mechanism, since these receptors may play a role in the clearance of endothelins from the circulation.22 In DOCA-salt hypertensive rats treated with bosentan, we detected this rise in immunoreactive ET-1 levels in plasma12 and an increase in ET-1 immunoreactive content and mRNA abundance (R. Larivière and E.L. Schiffrin, unpublished observations, 1994), which could also contribute to the increase in plasma endothelin immunoreactivity after bosentan treatment. WKY in the current study exhibited a trend toward an increase in plasma concentrations of immunoreactive ET-1 after bosentan treatment, but the elevation in concentration was not statistically significant. In SHR there was no change in the plasma levels of ET-1, which together with our previous findings of slightly lower levels of vascular immunoreactive ET-111 and ET-1 mRNA in SHR20 may suggest that SHR are more resistant to stimuli that enhance ET-1 expression. This remains to be confirmed by further experiments. Another possible explanation could be that decreased occupancy of ETB receptors in the endothelium caused by reduced vascular production of ET-1 could result in more effective buffering of rises in endothelin concentration in plasma by these receptors, considering their potential role in the clearance of endothelin,22 and this despite ETB receptor blockade by bosentan.
In the present study as in previous ones,14 15 SHR were lighter than age-matched control WKY, which introduces a confounding factor in comparisons of blood vessel structure. We have recently examined the correlation of body weight with the structure of conduit and resistance vessels in normotensive rats (as described in “Analysis of Data”). We observed that when the body weight of rats was 350 g or higher, the wet weights of segments of identical length of aorta or of the complete mesenteric arterial bed did not change. Lumen diameter, the media width, and the ratio of media width to lumen diameter of small arteries exhibited very small differences (<5%) attributable to differences in the body weight of the rats. Thus, no corrections were necessary for any of these parameters in the present study because the body weight of all SHR was approximately 350 g and that of WKY was 460 g. In contrast, the cross-sectional area of the media of small arteries correlated with the square root of the ratio between 350 and the body weight of rats. Thus, for WKY weighing 460 g, the media cross section should be normalized for comparison with SHR weighing 350 g by multiplying by the empirical factor (350/460)1/2=0.87. When cross-sectional areas of the media of small arteries of WKY were thus corrected, the cross sections of the media of coronary, mesenteric, and femoral small arteries remained similar in SHR and WKY, indicating that the predominant finding in small arteries in these vascular beds is remodeling (vessels with smaller diameter and no wall or media hypertrophy). In contrast, renal cortical small arteries of SHR exhibited significantly greater cross-sectional areas of the media than WKY arteries after the latter were corrected for body weight, indicating that in these SHR arteries, vascular hypertrophy may be predominant. This agrees with the finding that there was no significant difference between the lumen diameters of renal small arteries of SHR and WKY.
In conclusion, our data establish that the administration of the combined ETA/ETB endothelin receptor antagonist bosentan for 4 weeks at a daily dose that effectively blocks both ETA and ETB receptors does not alter blood pressure or the structure of blood vessels of adult SHR. This suggests that endothelins do not participate in the maintenance of elevated blood pressure in adult SHR. This study also shows that small arteries from the coronary, renal, mesenteric, and femoral circulations of SHR exhibit essentially similar alterations, with vascular hypertrophy and remodeling, the mechanism of which does not appear to involve endothelins.
This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension and by a grant from the Fondation des maladies du coeur du Québec. The authors are grateful for the technical help of André Turgeon and Micheline Vachon, the collaboration of Dr Li Yuan Deng, and the secretarial help of Angie Poliseno.
Reprint requests to Ernesto L. Schiffrin, MD, PhD, Experimental Hypertension Laboratory, Clinical Research Institute of Montréal, 110 Pine Ave W, Montréal, Quebec H2W 1R7, Canada.
- Received August 29, 1994.
- Revision received October 12, 1994.
- Accepted December 9, 1994.
Bobik A, Grooms A, Millar JA, Mitchell A, Grinpukel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol. 1990;258:C408-C415.
Batra VK, McNeil JR, Xu Y, Wilson TW, Gopalakrishnan V. ETB receptors on aortic smooth muscle cells of spontaneously hypertensive rats. Am J Physiol. 1993;264:C479-C484.
De Nucci G, Thomas R, D’Orléans-Juste P, Antunes E, Walder C, Warner TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A. 1988;85:9797-9800.
Deng LY, Schiffrin EL. Effects of endothelin on small arteries of DOCA-salt hypertensive rats. Am J Physiol. 1992;262:H1782-H1787.
Nguyen PV, Parent A, Deng LY, Flückiger JP, Thibault G, Schiffrin EL. Endothelin vascular receptors and responses in DOCA-salt hypertensive rats. Hypertension. 1992;19(suppl II):II-98-II-104.
Larivière R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension. 1993;21:294-300.
Li JS, Larivière R, Schiffrin EL. Effect of a nonselective endothelin antagonist on vascular remodeling in DOCA-salt hypertensive rats. Hypertension. 1994;24:183-188.
Dohi Y, Luscher TF. Endothelin in hypertensive small arteries: intraluminal and extraluminal dysfunction. Hypertension. 1991;18:543-549.
Deng LY, Schiffrin EL. Effects of endothelin-1 and vasopressin on small arteries of spontaneously hypertensive rats. Am J Hypertens. 1992;5:817-822.
Clozel M, Breu V, Gray GA, Kalina B, Löffler B-M, Burri K, Cassal J-M, Hirth G, Muller M, Neidhart W, Ramuz H. Pharmacological characterization of bosentan, a new potent orally active non-peptide endothelin receptor antagonist. J Pharmacol Exp Ther. 1994;270:228-235.
Larivière R, Li J-S, Sventek P, Thibault G, Schiffrin EL. Endothelin-1 gene expression and vascular hypertrophy during development of DOCA-salt hypertension in comparison to spontaneously hypertensive rats. Clin Exp Physiol Pharmacol. 1994;21(suppl 1):S64. Abstract.