Angiotensin II Increases Tissue Endothelin and Induces Vascular Hypertrophy
Reversal by ETA-Receptor Antagonist
Background In vitro studies on vascular smooth muscle cells suggest that endothelin has a stimulating effect on cellular proliferation. This study was designed to determine the endogenous effect of endothelin on angiotensin II–induced hypertrophy of small arteries in vivo.
Methods and Results Two weeks of angiotensin II administration (200 ng·kg−1·min−1) increased media thickness, media/lumen ratio, and cross-sectional area of basilar and small mesenteric arteries, confirming the proliferative properties of angiotensin II. The tissue levels of endothelin-1 were elevated in mesenteric arteries after angiotensin II administration. The administration of the selective and specific ETA-receptor antagonist LU135252 (50 mg·kg−1·d−1) in combination with angiotensin II prevented the changes of vascular geometry and partially reduced the increase in blood pressure induced by angiotensin II. Indeed, part of the effect on the vascular structure of the endothelin-receptor antagonist seemed pressure-independent.
Conclusions Our results therefore demonstrate that angiotensin II increases the production of endothelin in the blood vessel wall that, via ETA receptors, mediates changes in vascular structure of the cerebral and mesenteric circulation. Endothelin antagonists may therefore be of value to reduce blood pressure and to prevent vascular structural changes in conditions of increased activity of the renin-angiotensin system.
The endothelium regulates vascular tone by releasing vasoactive substances such as NO and ET-1.1 In addition, it is becoming increasingly clear that these endothelium-derived mediators can also affect the migration and proliferation of VSMCs and could therefore be involved in the modification of vascular structure observed in hypertension. As such, ET-1 has proved to be a mitogen2 3 and to induce protein synthesis4 in cultured VSMCs. Furthermore, in DOCA-salt hypertension, in which endothelial expression of ET-1 is enhanced,5 ET-receptor blockade reduces vascular hypertrophy.6
In hypertension, resistance arteries undergo structural changes as an adaptation to increased wall tension.7 These morphological changes may protect the microcirculation against the blood pressure rise, but they could also contribute to the maintenance of hypertension8 and, particularly in the cerebral circulation, to vascular complications. Alterations of small-artery structure may be mediated by eutrophic or hypertrophic vascular remodeling or a combination of the two.8 9 Because chronic administration of Ang II induces hypertrophic remodeling,10 we used this model to study the influence of ET on hypertrophy of small vessels in vivo. Blockade of ETA receptors was used to determine the role of endogenous ET on Ang II–induced proliferation. We hypothesized that a reduced vascular hypertrophy should be observed when an ETA-receptor antagonist is administered with Ang II, because Ang II stimulates the expression and release of ET by endothelial cells.11 12 This would, in turn, confirm the proliferative properties of ET in vivo and its involvement in conditions of increased activity of the renin-angiotensin system.
Nine-week-old Wistar Kyoto rats were purchased from IFFA CREDO (L’Arbesle, France). Twenty-eight rats (seven per group) were treated for 2 weeks with either Ang II, LU135252, or a combination of Ang II plus LU135252. Seven untreated rats served as controls. Ang II was administered by subcutaneously implanted osmotic pumps (model 2002, Alzet Corp) delivering 200 ng·kg−1·min−1 for 14 days. The ETA-receptor antagonist LU135252 was administered in the food at an average dose of 46±1 mg·kg−1·d−1 in the LU135252 group and 57±4 mg·kg−1·d−1 in the Ang II plus LU135252 group. Before and at the end of treatment, rats were weighed and systolic blood pressure and heart rate were determined by the tail-cuff method (model LE 5000, Letica). All procedures were approved by the Commission for Animal Research of the Canton of Bern, Switzerland.
Animals were anesthetized (thiopental 50 mg/kg IP), and the basilar artery and a segment of the fourth branch of the mesenteric arterial bed (closest segment to the ileum) were isolated under a dissecting microscope in cold (4°C) Krebs solution of the following composition (in mmol/L; control solution): NaCl 118.6, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.1, edetate calcium disodium 0.026, and glucose 10.1. Arteries were then mounted on and sutured to two small glass cannulas positioned in a vessel chamber (Living Systems Instrumentation) and superfused with control solution (37°C; 95% O2/5% CO2). The perfusion chamber was positioned on the stage of an inverted microscope (Nikon, TSM-F), and the amplified image was transmitted to a monitor and a video dimension analyzer (V91, Living Systems Instrumentation), allowing for measurements of lumen diameter and media thickness. Longitudinal stretch was controlled by adjustment of vessel length to a value slightly superior to the one required to produce a small bending of the vessel at 35 mm Hg of perfusion pressure.
Mesenteric arteries were allowed to equilibrate for 60 minutes and were perfused with control solution containing 1% BSA at constant perfusion pressure (30 mm Hg; Reference 1313 ); resting lumen diameter and media thickness were recorded. Basilar arteries were equilibrated for 60 minutes in a calcium-free control solution to prevent myogenic tone. Perfusion pressure was then increased from 25 to 55 mm Hg in 10 mm Hg steps, and the efferent pressure was adjusted to maintain a constant flow. Vascular structure was determined at each pressure.
LU135252, the active isomer of LU127043, is a selective ETA-receptor antagonist (ratio of affinities for ETA over ETB receptors, 161; Reference 1414 ). Its specificity has been evaluated by radioligand binding assays, and it did not demonstrate any interaction with several receptors, including the rat AT1 receptor (Dr M. Kirchengast, Knoll AG, personal communication). To confirm this, we compared Ang II–mediated contractions in the absence and presence of LU135252 preincubation in aortic rings. Contractions to 10−7 mol/L Ang II, expressed as a percentage of KCl contraction, were as follows: control, 23±3; LU135252 10−6 mol/L, 25±1; and LU135252 10−5 mol/L, 22±2 (n=4; P=NS versus control). Furthermore, contractions to Ang II of mesenteric arteries harvested from rats treated long-term with Ang II (24±5%, n=7) were not different if LU135252 was administered simultaneously (27±7%, n=7).
In separate experiments, 7 to 9 rats were chronically treated as described, and first- to fourth-order branches of the mesenteric arterial tree were dissected free from surrounding tissue for measurement of ET-1 tissue levels. The measurements were performed in a blinded fashion. Tissues were extracted according to a modification of the procedure of Hisaki et al.14A Vessels were weighed and homogenized in a polytron for 60 seconds in 2 mL of ice-cold chloroform:methanol 2:1 containing 1 mmol/L N-ethylmaleimide and 0.1% trifluoroacetic acid. Homogenates were left overnight at 4°C, then 0.8 mL of sterile distilled water was added. The mixture was vortexed and centrifuged at 4000 rpm for 15 minutes, and the supernatant was removed. Aliquots of the extract (1 mL) were diluted with 9 mL of 4% acetic acid and then extracted as described for plasma.15 Eluates were dried in a speed-vac and reconstituted in working assay buffer for radioimmunoassay. Overall recovery for ET-1 added to chloroform/methanol homogenates of vessels and taken through all extraction steps was 65±3%, with interassay and intra-assay coefficients of 5.6% and 10%, respectively (n=6). Plasma ET levels were determined as described.15
Values are mean±SEM. The CSA, growth index, and remodeling index were calculated as described.8 16 Because CSA does not change with changes in pressure, a mean of the values obtained at four different pressures (see above) was calculated for the basilar artery. The distensibility of the basilar artery is expressed as micrometer changes per mm Hg of pressure increase and represents the slope of the pressure–lumen diameter curve. Statistical evaluation was done by a one-way ANOVA with Bonferroni’s correction for multiple comparisons17 or by one-sample analysis (growth index, Fig 1⇓). The contrasts selected a priori for the ANOVA were Ang II and LU135252 compared with the control group, and Ang II plus LU135252 compared with Ang II alone. Pearson’s correlation coefficients were calculated by linear regression. A value of P<.05 was considered significant.
Body weight was reduced in the Ang II group compared with controls, whereas it was normal in all other groups, including the one treated with Ang II in combination with the ETA-receptor antagonist LU135252 (Table 1⇓). Chronic administration of Ang II significantly increased systolic blood pressure (Table 1⇓, Fig 1A⇑). Although LU135252 had no depressor effect on its own, it reduced the Ang II–induced increase in blood pressure (P<.05). Heart rate was not significantly modified by any chronic treatment, although there was a tendency for Ang II to increase it.
In basilar and small mesenteric arteries, Ang II administration increased media thickness and media/lumen ratio without modifying lumen diameter (Table 2⇓). The CSA and growth index were also significantly increased by Ang II (Table 2⇓, Fig 1B⇑). Ang II administration nearly doubled tissue ET-1 levels (P<.001; Fig 2⇓), whereas plasma levels of the peptide showed only a tendency to increase (from 2.6±0.1 to 4.1±0.5 pg/mL; P=NS).
The ETA-receptor antagonist LU135252 on its own had no effect on structure of mesenteric arteries compared with control vessels. In the basilar artery, LU135252 reduced media thickness and CSA, leading to a negative growth index (Fig 1B⇑). All structural vascular changes induced by the administration of Ang II for 2 weeks were prevented in both vascular beds by the concomitant oral administration of LU135252 (Table 2⇑, Fig 1B⇑). Compared with chronic Ang II administration alone (which nearly doubled mesenteric artery ET-1 levels; Fig 2⇑), LU135252 added to the treatment reduced tissue ET-1 levels (P<.05 versus Ang II alone; Fig 2⇑). On its own, LU135252 had a tendency to reduce tissue ET-1 levels (P=NS). In contrast, plasma levels of ET-1 were further increased by LU135252 from 2.6±0.1 pg/mL in the placebo group to 6.7±0.4 pg/mL (P<.05) and from 4.1±0.5 pg/mL in the Ang II group to 8.0±0.7 pg/mL in the Ang II plus LU135252 group (P<.05).
To confirm that the effect of LU135252 on Ang II–induced hypertrophy is not due solely to its hypotensive effect, the relationship between CSA and systolic blood pressure was constructed (Fig 3A⇓). The Ang II group that was also treated with LU135252 was significantly dissociated from the linear regression constructed with the control, Ang II, and LU135252 groups (P<.005 for basilar and mesenteric arteries; Fig 3A⇓).
There were strong positive correlations between systolic blood pressure and media/lumen ratio in both basilar (r=.75, P<.001) and small mesenteric arteries (r=.81, P<.0001; Fig 3B⇑). The distensibility of the basilar artery, as determined by the pressure–lumen diameter curves, was similar in all groups (slope ≈2 μm/mm Hg, data not shown), implying that the modifications of vascular structure could not be accounted for by an increased stiffness of the vessel wall.
In this study, we investigated the contribution of ET to Ang II–induced vascular hypertrophy in vivo. The involvement of endogenous ET in Ang II–induced hypertrophy was determined by the chronic administration of an ETA antagonist LU135252 and by measuring local ET tissue concentrations in the blood vessel wall. Our results demonstrate that Ang II increases vascular ET-1 tissue levels, which in turn mediate a major part of Ang II–stimulated vascular growth and hypertension in vivo.
Chronic administration of Ang II produced thickening of the arterial media as well as an increased media/lumen ratio of basilar and small mesenteric arteries. This alteration of vascular geometry was not accompanied by rearrangement of vascular tissue around a smaller lumen (calculated remodeling index, 8%), a process known as eutrophic remodeling,8 18 but rather by media hypertrophy, as shown by the growth index. Our results therefore confirm those previously reported10 and justify the use of this model to study vascular hypertrophy. Although we did not evaluate whether the increased CSA was secondary to hypertrophy or hyperplasia, a previous study has suggested that the smooth muscle cells of resistance arteries were hypertrophied in this model.8 In addition, this study for the first time demonstrates that Ang II is able to markedly increase vascular ET-1 tissue concentrations, whereas the increase in plasma levels of the peptide was less striking. These results are in line with previous in vitro studies showing that Ang II stimulates ET messenger RNA expression in cultured endothelial cells,11 12 and they extend these findings to in vivo conditions. Furthermore, these results demonstrate that ET is a paracrine local system and that its interaction with the renin-angiotensin system is most striking in the vessel wall rather than in plasma.
The selective ETA-receptor antagonist LU135252 prevented the Ang II–induced hypertrophy of small mesenteric and cerebral arteries. This is, to the best of our knowledge, the first direct evidence of an involvement of ET in Ang II–induced vascular hypertrophy in vivo. This conclusion is strengthened by markedly elevated ET-1 tissue levels in small mesenteric arteries after chronic administration of Ang II and explains the efficacy of an ETA-receptor antagonist to block Ang II–mediated vascular responses. It was of interest to note that LU135252 reduced tissue concentrations of ET-1 but increased plasma levels of the peptide in Ang II–treated animals. The increase in plasma levels of ET-1 during receptor blockade is known and is probably related to displacement of ET-1 from its receptor as well as reduced clearance of the peptide. The fact that LU135252 reduced tissue levels of ET-1 was surprising but may be related to displacement of the peptide from the tissues to the circulation. Alternatively, LU135252 may directly interfere with the production of ET in vascular tissue through its ETA-receptor interaction.
A study in two models of renovascular hypertension failed to demonstrate beneficial effects of an ETA/B-receptor antagonist on vascular structure and blood pressure.19 Because the renin-angiotensin system is involved in this model, the discrepancy with the present study is difficult to reconcile. It is likely that during chronic renovascular hypertension, the renin-angiotensin system is less activated and hence tissue ET levels are no longer increased, or possibly, the time at which treatment is initiated may be of importance. In contrast, one previous study in DOCA-salt hypertensive rats reported a decreased proliferation of the vascular wall with an ETA/B-receptor antagonist.6 However, in the latter model, it is unlikely that Ang II was the endogenous stimulus for increased synthesis of ET, because the activity of the renin-angiotensin system was negligible. Thus, ET may be an important mediator of vascular hypertrophy in hypertension, but the stimulus for its release differs in Ang II–induced and DOCA-salt hypertension.
To our surprise, administration of an ETA-receptor antagonist alone produced relative atrophy (decreased media thickness and CSA, negative growth index) of the basilar artery but not of mesenteric arteries. The cerebral arteries may therefore be under tonic proliferative influence from ET to maintain vascular structure, whereas this is not the case in a peripheral vascular bed.
It is also of interest to note that LU135252 prevented a good part of Ang II–induced hypertension. In isolated small arteries, Ang II–induced vasoconstriction is partly mediated by ET.11 20 Hence, this could explain the antihypertensive effectiveness of LU135252 in this model, which is in contrast to the relative ineffectiveness of ET-receptor antagonists in other models of hypertension, such as the spontaneously hypertensive rat.6 21 Endothelin-receptor antagonists may therefore be more effective in conditions associated with activation of the renin-angiotensin system. In that respect, the literature is conflicting, with one study showing no hypotensive effect of an ET-receptor antagonist in two-kidney, one-clip and one-kidney, one-clip hypertension,19 whereas another study showed a blood pressure reduction in subtotal nephrectomy hypertension.22
To support our conclusion, it was crucial to confirm that LU135252 does not bind to AT1 receptors (see “Methods”). Indeed, radioligand studies and pharmacological experiments with isolated vessels have excluded any interference of LU135252 with AT1 receptors. Furthermore, increased vascular ET-1 levels produced by Ang II suggest an interaction between the two pathways in vivo, independently of LU135252.
Hemodynamic parameters have an impact on vascular structure,7 8 and this is confirmed by the relationship between systolic blood pressure and media/lumen ratio in the present study. Hence, it may be argued that the antihypertensive effect of LU135252 could be responsible for normalization of vascular hypertrophy. However, Fig 3A⇑ demonstrates that LU135252 must have direct pressure-independent effects on hypertrophy. Furthermore, in a study using the same Ang II regimen, hydralazine lowered blood pressure but did not modify Ang II–induced vascular hypertrophy,10 suggesting that part of the proliferative effect of Ang II is pressure-independent. Hence, we postulate that stimulation of vascular ET production represents a pressure-independent mechanism of vascular hypertrophy.
In conclusion, vascular hypertrophy and the increase in blood pressure induced by Ang II in vivo is mediated at least in part by an increased production of endogenous ET, which then activates ETA receptors to produce the observed changes on the cardiovascular system. Endothelin-receptor antagonists may therefore represent an alternative approach for the treatment of cardiovascular diseases associated with an increased activity of the renin-angiotensin system.
Selected Abbreviations and Acronyms
|VSMC||=||vascular smooth muscle cell|
LU135252 was kindly provided by Dr M. Kirchengast, Knoll AG, Ludwigshafen, Germany. This work was supported by a grant from the Swiss National Research Foundation (grant 32-32541.91). Pierre Moreau holds a fellowship from the Medical Research Council of Canada, and Livius V. d’Uscio receives a stipend from the Intermedia Foundation, Bern, Switzerland. Matthias Barton was supported by the Deutsche Forschungsgemeinschaft (Ba 1543-1).
- Received December 9, 1996.
- Revision received March 25, 1997.
- Accepted March 26, 1997.
- Copyright © 1997 by American Heart Association
Lüscher TF. Local relaxant and constricting factors in the vessel wall. In: Swales JD, ed. Textbook of Hypertension. London, UK: Blackwell Scientific Publications; 1994:145-159.
Dubin D, Pratt RE, Cooke JP, Dzau VJ. Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J Vasc Med Biol. 1989;1:13-17.
Chua BHL, Krebs CJ, Chua CC, Diglio CA. Endothelin stimulates protein synthesis in smooth muscle cells. Am J Physiol. 1992;262:E412-E416.
Li JS, Larivière R, Schiffrin EL. Effect of a nonselective endothelin antagonist on vascular remodeling in deoxycorticosterone acetate-salt hypertensive rats: evidence for a role of endothelin in vascular hypertrophy. Hypertension. 1994;24:183-188.
Heistad DD, Baumbach GL. Cerebral vascular changes during chronic hypertension: good guys and bad guys. J Hypertens. 1992;10(suppl 7):S71-S75.
Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany MJ. Small artery structure in hypertension: dual process of remodeling and growth. Hypertension. 1993;21:391-397.
Mulvany MJ, Baumbach GL, Aalkjaer C, Heagerty AM, Korsgaard N, Schiffrin EL, Heistad DD. Vascular remodeling. Hypertension. 1996;28:505-506. Letter.
Griffin SA, Brown WCB, Macpherson F, McGrawth JC, Wilson VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension. 1991;17:626-635.
Dohi Y, Hahn AWA, Boulanger CM, Bühler FR, Lüscher TF. Endothelin stimulated by angiotensin II augments contractility of spontaneously hypertensive rat resistance arteries. Hypertension. 1992;19:131-137.
Takase H, Moreau P, Küng CF, Nava E, Lüscher TF. Antihypertensive therapy improves the endothelial function of resistance arteries in nitric oxide deficient hypertension: effect of verapamil and trandolapril. Hypertension. 1996;27:25-31.
Raschack M, Unger L, Riechers H, Klinge D. Receptor selectivity of endothelin antagonists and prevention of vasoconstriction and endothelin-induced sudden death. J Cardiovasc Pharmacol. 1995;26(suppl 3):S397-S399.
Hisaki K, Matsumura Y, Maekawa H, Fujita K, Takaoka M, Morimoto S. Conversion of big ET-1 in the rat lung: role of phosphoramidon-sensitive endothelin-1-converting enzyme. Am J Physiol.. 1994;266:H422-H428.
Moreau P, Takase H, Küng CF, Shaw S, Lüscher TF. Chronic endothelin-receptor blockade in chronic nitric oxide deficient hypertension. Hypertension. 1997;29:763-769.
Moreau P, Takase H, Küng CF, van Rooijen M-M, Schaffner T, Lüscher TF. Structure and function of the rat basilar artery during chronic nitric oxide synthase inhibition. Stroke. 1995;26:1922-1929.
Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9.
Li JS, Knafo L, Turgeon A, Garcia R, Schiffrin EL. Effect of endothelin antagonism on blood pressure and vascular structure in renovascular hypertensive rats. Am J Physiol. 1996;40:H88-H93.
Chen L, McNeill JR, Wilson TW, Gopalakrishnan V. Heterogeneity in vascular smooth muscle responsiveness to angiotensin II: role of endothelin. Hypertension. 1995;26:83-88.
Li J-S, Schiffrin EL. Effect of chronic treatment of adult spontaneously hypertensive rats with an endothelin receptor antagonist. Hypertension. 1995;25:495-500.
Potter GS, Melaragno MG, Johnson RJ, Fink GD. Evidence for a role of endothelin in the hypertension of experimental chronic renal failure. Hypertension. 1996;28:542. Abstract.