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(Hypertension. 1995;25:769-773.)
© 1995 American Heart Association, Inc.

Articles

Deoxycorticosterone Acetate Plus Salt Induces Overexpression of Vascular Endothelin-1 and Severe Vascular Hypertrophy in Spontaneously Hypertensive Rats

Ernesto L. Schiffrin; Richard Larivière; Jin S. Li; Pavol Sventek; Rhian M. Touyz

From the Medical Research Council of Canada Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal (Québec, Canada).

	Abstract

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Abstract Endothelin-1 gene expression is enhanced in the aorta and mesenteric arteries, and possibly other vessels, of deoxycorticosterone acetate (DOCA)–salt hypertensive rats. In contrast, endothelin-1 gene expression is normal or reduced in spontaneously hypertensive rats (SHR). Severe vascular hypertrophy is present in DOCA-salt hypertensive rats but not in SHR. In this study we investigated whether treatment of SHR with DOCA and salt would result in enhanced endothelin-1 expression and at the same time in severe vascular hypertrophy. Increased abundance of endothelin-1 mRNA was found in the aorta and the mesenteric arterial bed of SHR treated simultaneously with DOCA and salt but not when rats were treated with either separately. The wet weight of the aorta and of the mesenteric arterial bed, media thickness, media cross-sectional area, and media-to-lumen ratio of mesenteric small arteries of DOCA- salt–treated SHR were exaggerated beyond what could be explained by the elevation of blood pressure, relative to SHR treated with salt or with DOCA, which did not overexpress vascular endothelin-1. In conclusion, SHR may exhibit enhanced expression of the endothelin-1 gene in blood vessels when treated with DOCA and salt, and associated with this there is severe vascular hypertrophy. These data support the hypothesis of a role of endothelin-1 in vascular hypertrophy.

Key Words: endothelins • aorta • mesenteric arteries • vascular resistance • hypertrophy • deoxycorticosterone • sodium, dietary

	Introduction

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Endothelin-1 is a potent vasoconstrictor^{1 2} and has accordingly been the subject of many studies attempting to determine its potential involvement in experimental or human hypertension. Despite the fact that in most studies normal circulating levels of endothelin-1 immunoreactivity have been reported in deoxycorticosterone acetate (DOCA)–salt hypertensive rats,^{3 4} immunoreactive endothelin-1 is increased in endothelial cells of the aorta and mesenteric arteries of rats with this form of experimental hypertension.⁵ Abundance of endothelin-1 mRNA is also significantly elevated in vessels of DOCA-salt hypertensive rats.⁶ In blood vessels from spontaneously hypertensive rats (SHR), the content of endothelin-1 is not increased and may even be lower than that found in Wistar-Kyoto (WKY) control rats.⁵ DOCA-salt hypertensive rats exhibit a more severe degree of vascular hypertrophy⁷ compared with SHR.⁸ We proposed that endothelin-1, which possesses hypertrophic and mitogenic properties,^{9 10 11} could play a role in the severe vascular hypertrophy present in DOCA-salt hypertensive rats, in addition to its vasoconstrictor action.

To investigate whether inducing the expression of the endothelin-1 gene in blood vessels of SHR would result in exaggerated vascular hypertrophy as found in DOCA-salt hypertension, we subjected adult SHR to treatment with DOCA, salt, or both. We then examined the abundance of transcripts of the endothelin-1 gene and the degree of vascular hypertrophy in conduit and small arteries.

	Methods

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Animal Experiments
The study protocol was approved by the Animal Care Committee of the Clinical Research Institute of Montreal and was carried out according to the recommendations of the Canadian Council of Animal Care. Rats were housed under conditions of constant humidity (60%) and temperature (22°C) and subjected to 12-hour light/dark cycles. To serve as a positive control in this study, DOCA-salt hypertension was induced in Sprague-Dawley rats by the method of Ormsbee and Ryan,¹² since DOCA-salt hypertensive rats were previously shown to exhibit vascular endothelin-1 overexpression.^{5 6} Five male Sprague-Dawley rats (Charles River Laboratories, St Constant, Quebec) were unilaterally nephrectomized under pentobarbital anesthesia (40 mg/kg) (Somnotol, MTS Pharmaceuticals). Silicone rubber impregnated with DOCA (Sigma Chemical Co) (200 mg per rat) was implanted subcutaneously. Rats were offered 1% saline to drink. Thirty-two SHR (Taconic Farms, Germantown, NY) aged 13 weeks were treated as follows (8 rats per group): (1) they had silicone rubber implanted without DOCA, (2) they had silicone rubber implanted without DOCA and were offered 1% saline to drink, (3) they received DOCA and were offered tap water to drink, or (4) they received both DOCA and 1% saline. Eight WKY control rats were also bought from Taconic Farms at 13 weeks of age. All rats were studied at 17 weeks of age. Systolic blood pressure was taken by the tail-cuff method, after warming and under slight restraint. Blood pressure was recorded on a Grass model 7 polygraph (Grass Medical Instruments) fitted with a 7-P8 preamplifier and a model PCPB photoelectric pulse sensor. The average of three pressure readings was recorded. On the day of the experiment the rats were killed by decapitation. Blood was collected into tubes containing potassium edetate for measurement of immunoreactive endothelin-1, centrifuged, and stored at -70°C until assayed. A 1.5-cm-long segment of thoracic aorta and the complete mesenteric vascular bed were removed and dissected free of fat. Tissues were then snap-frozen in liquid nitrogen and stored at -70°C until extraction of total RNA was performed.

Northern Analysis
We extracted total RNA from frozen tissues by a guanidine isothiocyanate-phenol-chloroform method.¹³ Total RNA samples (20 µg) were denatured in 1x running buffer (20 mmol/L MOPS [pH 7.0], 6 mmol/L sodium acetate, 1 mmol/L EDTA), 6% formaldehyde, and 50% formamide for 15 minutes at 65°C. RNA samples were run on a 1.0% agarose gel containing 1x running buffer for 4 to 5 hours. The samples were transferred from the gel to a nylon membrane (Hybond-N; Amersham) by capillary action with 3 mol/L NaCl and 0.3 mol/L sodium citrate (20x SSC). After blotting, the membranes were dried by baking at 80°C for 2 hours. The locations of the 18S and 28S rRNA species were revealed by staining with 0.02% methylene blue in 0.3 mol/L sodium acetate (pH 5.5). Membranes were prehybridized at 60°C for 2 hours (42°C for the ³²P-labeled oligonucleotide probe for the 18S rRNA) in 400 mmol/L sodium phosphate buffer (pH 7.2) containing 5% SDS, 1 mmol/L EDTA, 0.1% bovine serum albumin, and 50% formamide. Hybridization with the ³²P-labeled probe was carried out for 18 to 20 hours at 60°C. The membranes were washed in 12.5 mmol/L NaCl and 0.1% SDS three times at 72°C for 20 minutes. The membranes were exposed to Reflection films (Dupont) with intensifying screens at -70°C for 6 days (2 to 4 hours for the 18S rRNA). The autoradiograms were analyzed with the use of a Bio-Rad imaging densitometer and MOLECULAR ANALYST software, version 1.1 (Bio-Rad Laboratories).

The rat endothelin-1 probe was prepared from rat lung RNA by reverse transcriptase–polymerase chain reaction (RT-PCR).⁶ A 319-bp rat preproendothelin-1 PCR product was obtained with the use of a 5' forward primer, 5'-CTAGGTCTAAGCGATCCTTG-3', and a 3' reverse primer, 5'-TTCTGGTCTCTGTAGAGTTC-3', located at nucleotides 266 to 285 and 565 to 584 of the coding sequence of the rat endothelin-1 cDNA, respectively.¹⁴ This PCR product was then cloned into pGEM-7zf(+) plasmid (Promega). The radiolabeled antisense riboprobe was prepared as previously described⁶ with the use of [³²P]UTP (800 Ci/mmol; Dupont). 18S rRNA was analyzed with a specific oligonucleotide probe (5'-CTTCCTCTAGATAGTCAAGTTCGACCGTCT-3')¹⁵ labeled with T4 polynucleotide kinase (Pharmacia) and [³²P]ATP (3000 Ci/mmol; Dupont). The ³²P-labeled probes were purified by chromatography with the use of a Sephadex G-50 column (Pharmacia) or NACS cartridges (Gibco-BRL) for the riboprobe and the oligonucleotide probe, respectively.

Preparation of Small Arteries
Mesenteric resistance arteries were studied as previously described.^{7 8} 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 1 mm from the intestine and approximately 2 mm in length was isolated. The vessel was mounted as a ring preparation on an isometric myograph (Living Systems Instrumentation). The dissection and mounting were performed in physiological salt solution (PSS) at room temperature. 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. 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. Media width was measured with a Leitz-Diavert inverted light microscope at a x320 magnification at 12 different sites along the wall, which were then averaged. The vessels were set to L₀, where L₀=0.9 L₁₀₀, and L₁₀₀ is the internal circumference the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. The media cross-sectional area was calculated from the lumen circumference and the media width of unstretched vessels, as previously described.^{16 17} The standardized media thickness (the parameter reported) was then obtained from media cross-sectional area, assuming a constant media volume, and from L₀. The lumen diameter was calculated as L₀/. Blood vessels were stimulated successively with 10 µmol/L norepinephrine, PSS in which NaCl was replaced by KCl on an equimolar basis (total KCl concentration, 124.7 mmol/L [KPSS]), and 10 µmol/L norepinephrine in KPSS to test reactivity of the small arteries.

Measurement of Immunoreactive Endothelin-1 in Plasma
Plasma endothelin-1 was measured by radioimmunoassay after extraction by passage through a C18 Sep-Pak cartridge, as previously described.¹⁸

Analysis of Data
Results are expressed as mean±SEM. Comparison of mean values was performed by ANOVA followed by the Student-Newman-Keuls test for multiple comparisons. Differences were considered significant at P<.05.

	Results

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Systolic Blood Pressure, Body Weight, and Plasma Endothelin-1
The body weights of rats of different groups were different at the end of the experiments (Table). SHR weighed less than age-matched WKY control rats. SHR receiving salt or DOCA attained slightly lower body weights, whereas DOCA-salt SHR gained less weight initially and then lost weight toward the end of treatment. As a result, their final body weight was 50% that of WKY rats. SHR treated with salt or DOCA did not exhibit higher systolic blood pressures than untreated SHR, whereas DOCA-salt SHR had higher blood pressures than other SHR groups. They also presented a more severe degree of left ventricular hypertrophy, as shown by the highest ratio of heart to body weight. There were only minor differences in plasma concentrations of immunoreactive endothelin-1 in the different groups of rats, with the highest levels found in DOCA-salt SHR, but these differences did not achieve statistical significance (F=2.304, P=.068; Table).

View this table: [in this window] [in a new window]   Table 1. Body Weight, Blood Pressure, Plasma Immunoreactive Endothelin-1, and Vascular Parameters

Vascular Endothelin-1 mRNA
Fig 1 shows a Northern blot of RNA extracted from the mesenteric arterial bed of all groups of rats, and Fig 2 shows results of densitometric analysis of three to four experiments. The 2.3-kb band corresponding to endothelin-1 mRNA exhibited greater intensity in lanes from vessels of DOCA-salt SHR and from a control group of DOCA-salt hypertensive rats (blood pressure, 198±5 mm Hg). No other group of rats demonstrated enhanced expression of the endothelin-1 gene in blood vessels. Identical results were obtained when RNA extracted from aorta was examined by Northern blot analysis. Similarly, the abundance of endothelin-1 mRNA was increased in the aorta of DOCA-salt SHR and, as expected,⁶ in DOCA-salt hypertensive rats (not shown).

View larger version (21K): [in this window] [in a new window]   Figure 1. Representative Northern blot of total RNA (20 µg per lane) extracted from the complete mesenteric arterial bed of Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), SHR drinking 1% saline (SHR+salt), SHR treated with deoxycorticosterone acetate (DOCA) (SHR+DOCA), SHR treated with DOCA and salt (SHR+D-s), and unilaterally nephrectomized Sprague-Dawley rats treated with DOCA and salt, that is, DOCA-salt hypertensive rats (D-s). The upper panel shows a single band of 2.3 kb corresponding to the rat endothelin-1 (ET-1) mRNA transcript. The analysis was done with the use of a specific 32P-labeled complementary RNA probe for rat preproET-1. The lower panel shows the band of the 18S rRNA on the same blots, which is obtained by hybridizing with a specific 32P-labeled oligonucleotide probe and used to normalize the abundance of ET-1 mRNA (see Fig 2). The DOCA-salt hypertensive rats were used as a positive control, since ET-1 mRNA is known to be increased in these.6 Their blood pressure after 3 weeks of hypertension was 198±5 mm Hg.

View larger version (14K): [in this window] [in a new window]   Figure 2. Bar graph shows the ratio of the optical density of the endothelin-1 (ET-1) mRNA to the 18S rRNA bands (mean±SEM) from three to four samples from RNA extracted from the mesenteric arterial bed of all groups of rats studied, analyzed as in Fig 1. Abbreviations are as in Fig 1 except SHR+D, SHR treated with DOCA; and DOCA-salt, unilaterally nephrectomized Sprague-Dawley rats treated with DOCA and salt (DOCA-salt hypertensive rats). *P<.01 vs other groups of rats.

Wet Weight of Aorta and Mesenteric Arteries
The wet weight of segments of the aorta and the whole mesenteric arterial bed was significantly increased in DOCA-salt SHR even though their body weight was half that of age-matched WKY rats and 70% that of untreated SHR (Table). Vascular hypertrophy of DOCA-salt SHR, as evaluated by vascular wet weight, was disproportionately greater (particularly the mesenteric arterial bed) than in the other groups, even though systolic blood pressure was only 30 mm Hg higher.

Structure of Small Arteries
The morphometric characteristics of small arteries of the mesenteric circulation of the different groups of rats studied after mounting on a wire myograph under standardized conditions are shown in the Table. In untreated SHR or in SHR treated with salt or DOCA, the lumen diameter was smaller and the media thickness of small arteries was greater than the corresponding parameters of WKY rats. The cross-sectional area of the media was similar. In DOCA-salt SHR, media thickness, ratio of media thickness to lumen diameter, and media cross-sectional area were significantly greater than in all other groups.

	Discussion

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In previous studies we proposed that endothelin-1 could be involved in the vascular hypertrophy found in DOCA-salt hypertensive rats independent of the effect of elevated blood pressure.^{5 6} We demonstrated in those studies that enhanced expression of the endothelin-1 gene could be detected in different vascular beds in this hypertensive model. We also showed that SHR did not exhibit overexpression of endothelin-1.⁵ Since SHR exhibit less vascular hypertrophy than DOCA-salt hypertensive rats,^{7 8} we suggested that the vascular overexpression of endothelin-1 in the latter hypertensive model could explain these differences. In this study we attempted to answer the following questions: (1) If SHR are treated with DOCA and salt, even though normally SHR exhibit normal or reduced expression of endothelin-1 in blood vessels compared with WKY control rats, will they overexpress the endothelin-1 gene? (2) If enhanced endothelin-1 gene expression is induced, will the severity of vascular hypertrophy in SHR be greater and beyond what can be attributed to the higher blood pressure found in this experimental paradigm? The answers to both questions from the current results are affirmative.

The mechanisms involved in vascular hypertrophy and remodeling in hypertension (the latter present in small arteries and characterized by a reduction in the diameter of the blood vessel¹⁹ ) are not well understood. Vascular hypertrophy may result from smooth muscle cell hyperplasia or hypertrophy and deposition of intercellular matrix.²⁰ The exact nature of the remodeling process is unknown. An altered arrangement of smooth muscle cells^{21 22} as well as changes in the compliance of resistance vessels,^{23 24} perhaps as a consequence of deposition of intercellular matrix, may play a role in its development. Few studies have examined the exact pathological changes occurring in DOCA-salt SHR, and this aspect was not addressed in this study. Sesoko et al²⁵ investigated the pathology of the kidney in malignant DOCA-salt SHR, after a longer evolution of hypertension (10 weeks) than we examined in the current study, when these rats had developed renal failure, and found wall thickening and obstruction of small arteries with hemorrhage and fibrinoid necrosis in the vascular wall. This could indicate that at this advanced period, activation of blood coagulation may occur. This phenomenon could play a primary role in enhanced expression of endothelin-1 by the endothelium or be secondary to increased vascular endothelin, with subsequent vasospasm, ischemia, and obstruction. Whether this occurs in other vascular beds and to what degree these changes are already present earlier, when blood pressure elevation is severe but has not reached the stage of malignant hypertension, as in our study, remain to be established. Endothelin-1 possesses mitogenic and hypertrophic properties^{9 10 11} and could thus play the hypertrophic role we are proposing in blood vessels when its vascular expression is enhanced, probably acting in concert with different growth factors. Recent studies have suggested that of the different endothelium-derived agents with growth-modulating potential, endothelin-1 may be the one that could contribute to a greater extent to vascular hypertrophy.²⁶ The exact nature of the process that is potentially triggered by endothelin-1 to result in vascular hypertrophy is complex and may include cell hypertrophy, hyperplasia, and increased deposition of intercellular matrix. The pathological characteristics of the process occurring in the vascular wall require further study and are beyond the scope of the present investigation. When DOCA-salt hypertensive rats, which (as mentioned) overexpress the endothelin-1 gene in blood vessels, are treated with the endothelin receptor antagonist bosentan, they develop a slightly smaller elevation of blood pressure but significantly less vascular hypertrophy.²⁷ Together with results recently obtained showing that long-term administration of the combined endothelin-A/endothelin-B receptor antagonist bosentan to DOCA-salt SHR will also result in slight lowering of blood pressure but important blunting of the excess vascular hypertrophy present in DOCA-salt SHR (J.S.L. and E.L.S., unpublished data, 1994), the present data provide further evidence of involvement of endothelin-1 overexpression in the exaggerated vascular hypertrophy found in this hypertensive animal model. However, a role of the higher blood pressure of DOCA-salt SHR in vascular hypertrophy in this model in comparison to the other SHR groups cannot be completely excluded, and further experiments are necessary to determine whether part of the excess vascular hypertrophy found in this experimental model is blood pressure dependent.

The mechanisms whereby DOCA together with salt induces enhanced expression of the endothelin-1 gene in unilaterally nephrectomized Sprague-Dawley DOCA-salt hypertensive rats^{5 6} and in DOCA-salt SHR (this study) are not well understood. We have shown that it takes 3 to 4 weeks for the enhancement of expression to become evident in Northern blot analysis of RNA from arteries from DOCA-salt hypertensive rats,²⁸ by which time these are hypertensive. However, two-kidney DOCA-salt rats, which are slightly less hypertensive, do not overexpress endothelin-1 in blood vessels. As mentioned above, the difference in blood pressure between untreated or DOCA- or salt-treated SHR and DOCA-salt SHR is small (30 mm Hg). This could suggest that elevated blood pressure per se is not a factor in the increased expression of endothelin-1 reported, although it may play a permissive role. Growth factors such as transforming growth factor–ß1,²⁹ whose expression is increased in DOCA-salt hypertension,³⁰ vasopressin,³¹ hemodynamic factors,³² or other factors undetermined as yet could be involved in the mechanisms that result in increased endothelin-1 expression under the combined influence of DOCA and salt. Activation of blood coagulation, as suggested by previous data regarding the pathology of blood vessels in more advanced stages of the evolution of hypertension than in this experimental model,²⁵ if occurring at this earlier stage, could also contribute to stimulation of endothelin-1 expression through the action of thrombin.^{1 33}

Together with our previous observations, the current study supports the hypothesis that endothelin-1 may play a role in blood pressure elevation and in vascular hypertrophy in some models of hypertension. Targeted disruption of the mouse endothelin-1 gene, which results in death of homozygotes, produces elevated blood pressure in heterozygotes.³⁴ This would seem to suggest that endothelin-1 cannot play a hypertensive role, in contrast to the results of our experiments. However, the gene "knock-out" experiments probably indicate that normally low concentrations of endothelin stimulate endothelial endothelin-B receptors, inducing secretion of nitric oxide and prostacyclin,³⁵ which contributes to the maintenance of normal vascular tone. When the endothelin-1 gene is disrupted and production of endothelin-1 is reduced in heterozygotes, the decreased production of nitric oxide and prostacyclin will result in less relaxation and consequently in vasoconstriction and elevation of blood pressure; this is similar to what occurs when inhibitors of nitric oxide synthase are administered, which also results in elevated blood pressure.³⁶ When endothelin-1 is overproduced in blood vessels, such as in DOCA-salt hypertensive rats,^{5 6} in the DOCA-salt SHR of the present study, and perhaps in other forms of hypertension, the excess endothelin-1 produced may reach vascular smooth muscle cells and result in vasoconstriction, vascular hypertrophy, and hypertension.

	Acknowledgments

 
This study was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension and by grants from the Fondation des maladies du coeur du Québec. Dr Touyz is supported by a fellowship of the Medical Research Council of Canada. Dr Larivière is a Scholar of the Fonds de recherche en santé du Québec. The authors thank André Turgeon for excellent technical help and Angie Poliseno for secretarial help.

	Footnotes

 
Reprint requests to Ernesto L. Schiffrin, MD, PhD, Experimental Hypertension Laboratory, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Québec, Canada H2W 1R7.

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