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Cardiovascular Research 2002 53(2):487-495; doi:10.1016/S0008-6363(01)00469-2
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Copyright © 2001, European Society of Cardiology

Chronic ETA receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice

Livius V d'Uscioa,1, Matthias Bartonc,1, Sidney Shawb and Thomas F Lüschera,*

aCardiovascular Research, Institute of Physiology, University of Zürich and Cardiology and University Hospital, Zürich, Switzerland
bDepartment of Clinical Research, University Hospital, Bern, Switzerland
cClinical Atherosclerosis and Genetic Research Laboratory, University Hospital Zurich, Zurich, Switzerland

* Corresponding author. University Hospital Zürich, Rämistrasse 100 CH-8091, Zürich, Switzerland. Tel.: +41-1-255-2121/2177; fax: +41-1-255-4251 cardiotfl{at}gmx.ch

Received 26 April 2001; accepted 28 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study investigated whether endothelial dysfunction occurs in mesenteric arteries of apoE-deficient mice and determined the role of endothelin (ET)-1, which is increased in human atherosclerosis, using an orally active endothelin ETA receptor antagonist. Methods: ApoE-deficient and C57BL/6J control mice were fed for 30 weeks with normal chow or high-fat Western-type diet alone or in combination with darusentan (LU135252; 50 mg/kg/day). Vasomotor reactivity of isolated small mesenteric arteries (I.D. 200–250 µm) was studied in vitro under perfused and pressurized conditions. Results: In both mouse strains, about one fourth of the endothelium-dependent relaxant response to acetylcholine was insensitive to inhibition by L-NAME and indomethacin. In mesenteric arteries of apoE-deficient mice on Western-type diet, increased intima-media thickness and levels of endothelin-1 protein were observed. In addition, NO-mediated endothelium-dependent relaxation to acetylcholine was reduced without affecting L-NAME/indomethacin insensitive relaxation and contractions to endothelin-1 and serotonin were enhanced. Treatment with darusentan normalized vascular structure, NO-mediated relaxation to acetylcholine and contractions to endothelin-1 and serotonin without affecting blood pressure or plasma cholesterol levels. Conclusions: Severe hypercholesterolemia in apoE-deficient mice is associated with attenuation of NO-mediated relaxation to acetylcholine and increased vascular endothelin-1 content. Chronic ETA receptor blockade may provide a new therapeutic approach to improve NO-mediated endothelium-dependent vasomotion in small arteries.

KEYWORDS Endothelins; Endothelial receptors; Endothelial function; Lipoproteins; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The vascular endothelium is a source of vasoactive substances such as nitric oxide (NO) [1] as well as endothelin-1 (ET-1) [2] that modulate vascular smooth muscle tone and proliferation. Endothelin (ET)-1 is a potent vasoconstrictor peptide [2] and its synthesis is stimulated by vasoactive factors implicated in atherogenesis [3]. ET-1 mediates contraction, cell proliferation [4,5], and vascular growth [6] via activation of ETA-receptors. In hypercholesterolemia an elevated plasma ET-1 concentration and enhanced coronary artery tissue ET-1 immunoreactivity were found [7,8].

Nitric oxide (NO) is a potent vasodilator and is formed from L-arginine by endothelial NO synthase [1]. NO as well as prostacyclin inhibit ET-1 production [9] and furthermore, reduce ET-1 induced vasoconstriction in vascular smooth muscle cells of small arteries [10,11]. This indicates that NO protects small arteries against contractions to ET-1 under physiological conditions. In hypercholesterolemia, a major risk factor predisposing to atherosclerosis, endothelial dysfunction is present before structural vascular changes occurs [12]. In addition, in epicardial coronary arteries both atherosclerotic lesions and endothelial dysfunction are present while intramyocardial coronary arteries exhibit only impairment of endothelium-dependent vasodilation [13–16].

It is unknown whether endothelial dysfunction occur in mesenteric arteries of apoE-deficient mice, which develop spontaneous hypercholesterolemia and aortic atherosclerosis that can be accelerated by a lipid-rich, Western-type diet [17,18]. Using a selective, orally active antagonist for ETA receptors, we investigated the role of ET-1 in small arteries in this mouse model of human atherosclerosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals and blood pressure
Male C57BL/6J (control) mice and homozygous apoE-deficient mice (C57BL/6J-ApoETm1Unc129) were obtained at the age of 4–5 weeks from The Jackson Laboratory (Bar Harbor, ME, USA). Housing facilities and all experimental protocols were approved by the local authorities for animal research (Kommission für Tierversuche des Kantons Zürich, Switzerland). To accelerate the development of spontaneous atherosclerotic lesions [19], control and apoE-deficient mice were fed a Western-type fat diet for 30 weeks (0.15% cholesterol and 42% of milk fat by weight, TD88137, Harlan Teklad, Madison, WI, USA) with or without the ETA receptor antagonist darusentan (50 mg/kg/day). In a separate protocol, apoE-deficient mice received normal chow diet with or without darusentan (50 mg/kg/day) for 30 weeks. Darusentan was mixed in powdered form to the diet and dosed as previously described [20,21]. Systolic blood pressure and heart rate were measured in conscious mice by a tail-cuff method using a custom-made pulse transducer (Model LE 5000, Letica, Barcelona, Spain).

2.2 Tissue harvesting
The mice were anesthetized (thiopental, 50 mg/kg body weight, i.p.) and sacrificed. A segment of the first branch of the mesenteric artery (closest to ileum; diameter: 200–250 µm) was isolated and placed in cold (4°C) modified Krebs–Ringer bicarbonate solution (in mmol/l: NaCl 118.6; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25.1; EDTA 0.026; glucose 10.1). Mesenteric arteries were dissected free from connective tissue under a microscope (Leica Wild M3C, Heerbrugg, Switzerland) and cut into rings of 4–5 mm in length.

2.3 Vascular function experimental set-up
Isolated mesenteric arteries were transferred to vessel chambers (Living Systems Instrumentation, Burlington, VT, USA), filled with Krebs–Ringer bicarbonate solution. The solutions circulating from a 250 ml reservoir at a flow-rate of 50 ml/min was aerated continuously with 95% O2–5% CO2 gas and kept at 37°C. The proximal and distal ends of the vessels were mounted and sutured onto two small glass microcannulas. The assembly was positioned in the vessel chamber and the perfusion pressure was set at 30 mmHg. In pilot studies, this was found to be optimal for contraction to norepinephrine as assessed by repeated exposures at various perfusions pressures (15–105 mmHg) using a transducer (Living Systems Instrumentation) connected to the afferent cannula. The intraluminal flow-rate was maintained at 0.2–0.4 ml/min. The perfusion chamber was positioned on the stage of an inverted microscope (Nikon, TSM-F, Japan) with a video camera (Burle Lancaster, PA, USA). The amplified image was transmitted to a monitor and a video dimension analyzer (V91, Living Systems Instrumentation), allowing for measurements and recording of lumen diameter and wall thickness.

2.4 Experimental protocols
Arteries were equilibrated for 60 min and perfused intraluminally with Krebs solution containing 1% BSA. Resting lumen diameter was recorded during perfusion with Krebs solution circulating from the reservoir. Between each protocol, the system was washed several times with Krebs solution and equilibrated for 30 min. Contractile responses to norepinephrine (10–10–10–5 mol/l), serotonin (10–10–10–5 mol/l) and ET-1 (10–11–3x10–8 mol/l) were obtained. Endothelium-dependent relaxations to acetylcholine (10–9–10–4 mol/l) after 30 min of preincubation with or without inhibitor of NO formation N{omega}-nitro-L-arginine methyl ester (L-NAME; 10–4 mol/l) or indomathacin (10–5 mol/l) alone or in combination of both were obtained after stabilization of half-maximal precontraction of mesenteric arteries with norepinephrine. For endothelium-independent relaxations, sodium nitroprusside (10–10–3x10–5 mol/l) was used.

2.5 Plasma cholesterol levels
Blood samples were obtained through puncture of the right ventricle. Blood was immediately transferred to a tube containing EDTA and centrifuged at 4°C for 10 min. Plasma was separated immediately at 4°C and kept at–80°C until assayed. Plasma LDL and HDL cholesterol were determined using a colorimetric-based assay on a Cobas Mira Plus® autoanalyzer (Roche Diagnostics, Basel, Switzerland).

2.6 Vascular ET-1 Protein Levels
Remaining mesenteric arterial tissue (n=4–8 per group) was dissected free, blotted dry, frozen in liquid nitrogen and kept at –80°C until assayed. Frozen tissue was crushed and homogenized as described [6]. Eluates were dried in a speed-vac and reconstituted in working assay buffer for the RIA. ET-1 was measured by RIA and the peptide was identified by reverse-phase HPLC and related to tissue weight [21].

2.7 Drugs
Acetylcholine hydrochloride, L-norepinephrine bitartrate, sodium nitroprusside dihydrate, 5-hydroxytryptamine (serotonin), indomethacin and L-NAME were from Sigma (St. Louis, MO, USA) and ET-1 was from Novabiochem (Läufelfingen, Switzerland). Darusentan (LU135252), the active (+)-isomer of LU127043, a non-peptide, selective ETA receptor antagonist was a gift from Knoll, Ludwigshafen, Germany.

2.8 Data analysis
Sensitivity to different drugs was expressed as negative logarithm of the concentration causing half-maximal relaxation or contraction (pD2 value). In addition, maximal contraction or relaxation (expressed as percentage of the decrease in the basal intraluminal diameter or of the increase in intraluminal diameter from the diameter obtained after precontraction, respectively) were determined for each individual concentration–response curve by non-linear regression analysis type sigmoid logistic using MATLAB® software as described elsewhere [20,22]. CSA was calculated as follows [23,24]: ({pi}/4)·[(I.D.+2·(intima+media)]2–(I.D.)2. Results are given as mean±S.E.M. In all experiments, n equals the number of mice. For multiple comparisons, results were analyzed with ANOVA followed by Bonferroni's correction. Pearson's correlation coefficient was calculated by linear regression analysis. A P value <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Mice characteristics
ApoE-deficient mice were normotensive and systolic blood pressure and heart rate did not differ between darusentan-treated apoE-deficient and control mice (Table 1). Body weight was increased in Western-diet fed apoE-deficient mice as compared with normal chow diet (P<0.05, Table 1) and was further increased in control mice on high-fat diet (P<0.05, Table 1). Heart weight was augmented in apoE-deficient mice on Western-type diet (P<0.05; Table 1). However, heart weight-to-body weight ratio was unchanged in apoE-deficient mice on Western-type diet as compared with normal chow diet (Table 1). Treatment with ETA receptor antagonist had no effect on these parameters in all groups of treated mice (Table 1).


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  		Table 1 Characteristics of apoE-deficient and C57BL/6J control mice after 30 weeks treatment

 
Plasma total cholesterol and LDL-cholesterol concentrations were elevated in apoE-deficient mice on normal diet as compared with C57BL/6J mice (P<0.05; Table 1). The Western-type diet further increased plasma cholesterol and LDL (P<0.05), but HDL was reduced (P<0.05; Table 1). Concomitant treatment with the ETA receptor antagonist had no effect on plasma cholesterol and LDL in apoE-deficient mice (Table 1) but increased HDL cholesterol levels in C57BL/6J mice (P<0.05; Table 1).

3.2 Vascular structure
In mesenteric arteries, lumen diameter did not differ in apoE-deficient and C57BL/6J mice (Table 2). Wall thickness and CSA were slightly increased only in apoE-deficient mice on Western-type fat-diet (P<0.05; Table 2) and were prevented by concomitant treatment with darusentan (P<0.05).


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  		Table 2 Morphological characteristics of mesenteric arteries from apoE-deficient and wild-type mice measured in perfused and pressurized conditions

 
3.3 Tissue ET-1 Levels
Tissue ET-1 content was increased in mesenteric arteries of apoE-deficient mice on Western-type diet as compared with apoE-deficient mice on normal diet or C57BL/6J mice (P<0.05; Fig. 1). Darusentan prevented the increase in tissue ET-1 levels in apoE-deficient mice to a greater degree as compared with C57BL/6J mice on Western-type diet (n=8, P<0.05, Fig. 1). Darusentan did not significantly affect vascular ET-1 levels in apoE-deficient mice on normal diet (n=4).


Figure 1
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  		Fig. 1 Tissue concentrations of ET-1 in small mesenteric arteries of apoE-deficient and C57BL/6J mice after 30 weeks treatment with Western type diet alone or in combination with the ETA receptor antagonist darusentan (n=4–8). For comparison, the results of normal fed apoE-deficient mice with or without darusentan are shown. *, P<0.05 vs. all other groups; {dagger}, P<0.05 vs. apoE-deficient mice on Western-type diet; #, P<0.05 vs. C57BL/6J mice (ANOVA+Bonferroni).

 
3.4 Endothelial function
Acetylcholine-induced relaxation was endothelium-dependent. The relaxations were impaired in apoE-deficient mice as compared to control mice on Western-type diet (P<0.05 vs. C57BL/6J; Fig. 2A; n=9). In contrast, the relaxations were unaltered in apoE-deficient mice fed normal diet (Fig. 2B; n=10). Incubation with L-NAME alone reduced maximal responses to acetylcholine to a comparable degree in apoE-deficient and C57BL/6J mice treated with and without darusentan (P<0.05 vs. without pretreatment; n=5–7; Fig. 2C and D). Preincubation with indomethacin in the presence or absence of L-NAME did not affect acetylcholine-induced relaxations (data not shown).


Figure 2
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  		Fig. 2 Endothelium-dependent relaxations to acetylcholine in small mesenteric arteries of apoE-deficient and control mice after 30 weeks treatment with Western-type diet (A and C) and normal diet (B and D) alone or in combination with the ETA receptor antagonist darusentan. Please note that relaxations to acetylcholine were reduced to the same extent in all groups in the presence of L-NAME (C and D). Results are shown as mean±S.E.M. (n=5–13 per group) and relaxations are expressed as percent of the increase in intraluminal diameter of the precontraction to norepinephrine.

 
Chronic treatment with darusentan, increased the L-NAME-sensitive portion of the response in apoE-deficient mice on Western-type diet (P<0.05; n=13; Fig. 2A). Maximal responses were inversely correlated with tissue ET-1 level in apoE-deficient mice on Western-type diet (r=–0.77; P<0.005; n=12) but not in C57BL/6J mice (P=0.95; n=13; NS).

Sensitivity but not maximal responses of endothelium-independent relaxations to sodium nitroprusside were slightly lower in arteries from apoE-deficient mice as compared to controls (P<0.05 vs. C57BL/6J, Fig. 3A) and was increased after darusentan (P<0.05 vs. apoE-deficient mice; Fig. 3A; n=5–9), without affecting the maximal response. In C57BL/6J mice on Western-type diet or apoE-deficient mice fed normal diet, no differences within treatment groups were observed (Fig. 3A and B).


Figure 3
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  		Fig. 3 Endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside in small mesenteric arteries of apoE-deficient and control mice after 30 weeks treatment with Western-type diet (A) and normal diet (B) alone or in combination with the ETA receptor antagonist darusentan. Results are shown as mean±S.E.M. (n=5–9 per group) and relaxations are expressed as percent of the increase in intraluminal diameter of the precontraction to norepinephrine.

 
3.5 Vascular contractility
Maximal contractions to ET-1 were unaffected by hypercholesterolemia, but the sensitivity of contractions to ET-1 was slightly increased in apoE-deficient mice on Western-type diet as compared with control mice (P<0.05; n=4; Fig. 4); this shift was normalized by darusentan (P<0.05; n=4; Fig. 4A). Concomitant treatment with darusentan had no effect on contractions to ET-1 in C57BL/6J mice on western-type diet or in apoE-deficient mice on normal diet (Fig. 4B).


Figure 4
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  		Fig. 4 Concentration-dependent contractions to ET-1 in small mesenteric arteries of apoE-deficient and control mice after 30 weeks treatment with Western-type diet (A) and normal diet (B) alone or in combination with the ETA receptor antagonist darusentan. Results are shown as mean±S.E.M. (n=4–7 per group) and contractions are expressed as percent decrease of basal intraluminal diameter.

 
Maximal contractions to serotonin were markedly higher in mesenteric arteries of apoE-deficient mice as compared to controls fed Western-type diet (P<0.05; Fig. 5A). The supersensitivity to serotonin was completely blocked by chronic treatment with darusentan (P<0.05; n=5–7; Fig. 5A).


Figure 5
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  		Fig. 5 Concentration-dependent contractions to serotonin (A) and norepinephrine (B) in small mesenteric arteries of apoE-deficient mice as compared with control C57BL/6J mice on Western-type diet. Results are shown as mean±S.E.M. (n=5–9 per group) and contractions are expressed as percent decrease of basal intraluminal diameter.

 
Contractions to norepinephrine were similar in apoE-deficient mice and C57BL/6J mice fed Western-type diet. Concomitant treatment with darusentan did not affect this response in apoE-deficient mice (Fig. 5B; n=5–7 per group).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This present study demonstrates that in small arteries of apoE-deficient mice fed Western-type diet tissue ET-1 content was elevated and was associated with increase in wall thickness and impairment of endothelium-dependent relaxation to acetylcholine. Chronic blockade of ETA receptors with darusentan normalized NO-mediated portion of the endothelium-dependent relaxation to acetylcholine and reduced increased ET-1 production and vascular reactivity to ET-1 without affecting systolic blood pressure or cholesterol levels.

Mice homozygous for the inactivated apolipoprotein E (apoE) gene provide a new model of human atherosclerosis. These mice develop spontaneous hypercholesterolemia and aortic atherosclerosis [17–19]. Heart rate and blood pressure were comparable between apoE-deficient and control mice on Western-type diet, but a significant decrease in body weight was found, while heart weight significantly increased. This is in line with the recent report by Hartley et al. [25]. Interestingly, our study showed that heart weight-to-body weight ratio was increased in apoE-deficient mice on normal diet and this increase was unaffected by Western-type diet. In addition, treatment with darusentan had no effect on these parameters in control and apoE-deficient mice irrespective of diet suggesting that ET-1 does not play a major role to these phenotypic alterations in apoE-deficient mice.

An impaired endothelium-dependent relaxation in response to acetylcholine has been observed in the aorta [26–29] and in coronary arteries of genetically altered hyperlipidemic mice [30]. Interestingly, although atherosclerosis primarily affects conduit arteries, endothelium-dependent relaxations are impaired not only in large arteries but also in small arteries of atherosclerotic animals and humans. In cremasteric vessels of cholesterol-fed rabbit cholinergic arteriolar vasodilation is defective [31]. In coronary arteries, hypercholesterolemia is associated with impaired endothelium-dependent relaxations to the agonists serotonin, bradykinin and histamine [13–16] indicating that the pathophysiological consequences of atherosclerosis may extend into small arteries also in humans. In our study, endothelium-dependent relaxations to acetylcholine were reduced in small arteries of apoE-deficient mice on Western-type diet. In contrast to the aorta [26,29], relaxation to acetylcholine in mesenteric arteries was inhibited only in part by L-NAME. The remaining portion of the response was not further attenuated by indomethacin, suggesting that an endothelium-dependent factor distinct from NO mediate part of relaxations to acetylcholine in mouse mesenteric arteries. Atherosclerosis had no effect on the L-NAME/indomethacin-insensitive portion of the response, contrasting a previous study that reported an augmentation of L-NAME/indomethacin-resistant relaxations to acetylcholine in renal arteries of hypercholesterolemic rabbits [32]. The discrepancy between these findings may be related to species differences and/or anatomic heterogeneity of vascular function. Furthermore, endothelium-dependent relaxations to acetylcholine were unaffected in the presence of indomethacin suggesting that endothelium-derived prostanoids do not play a major role in endothelial dysfunction. This is further confirmed by a recent study showing that no change in cyclic adenosine monophosphate occurred in the aorta of apoE-deficient mice [29].

Interestingly, chronic treatment with ETA receptor antagonist darusentan normalized impaired endothelium-dependent and -independent relaxations in small mesenteric arteries. However, only the L-NAME-sensitive portion of the response to acetylcholine was increased indicating that the improvement of vascular reactivity was due to increased bioactivity of NO. In line with these observations, we recently reported that plasma nitrate levels, an indicator of NO activity in vivo, are increased in darusentan-treated apoE-deficient mice [26]. Possible mechanisms may include up-regulation of endothelial NO synthase protein, as has been reported after chronic ETA blockade in hypercholesterolemic pigs [33,34]. In addition, the improvement in endothelium-dependent relaxations also appears to be related to an improved sensitivity of vascular smooth muscle cells to NO, as illustrated by the results obtained with the NO donor SNP.

ET-1 synthesis is stimulated by vasoactive factors and cytokines implicated in hypercholesterolemia [3]. ET-1 also stimulates cell proliferation and acts as a co-mitogen for vascular smooth muscle cells with other growth factors [6,35], suggesting that ET-1 contribute to structural changes through activation of multiple pathways. Importantly, ET-1 induces vascular hypertrophy is independent of systolic blood pressure [6,21] and in addition, ETA receptors promote the early inflammatory phase of atherosclerosis [36]. In our study, vascular ET-1 peptide content and contractions to ET-1 were increased in small mesenteric arteries of apoE-deficient mice as compared with control animals on Western-type diet. Increased contractions to ET-1 may be explained by the activation of signal transduction pathways of ET-receptors. In addition, we previously have shown that ETA receptor density in apoE-deficient mice aortas was increased [26]. The impairment of relaxation to acetylcholine was inversely related to vascular tissue ET-1 peptide content. Indeed, in apoE-deficient mice on normal diet neither endothelial dysfunction nor elevated tissue ET-1 content was found, despite a slight increase in plasma cholesterol levels. These observations strongly support a role for local ET-1 as an early participant in hypercholesterolemia and marker for endothelial dysfunction in animals and humans [7,8,33].

Interestingly, the ETA receptor antagonist darusentan markedly reduced tissue ET-1 levels and prevent structural changes in small mesenteric arteries, indicating that endogenous ET-1 contributes to endothelial dysfunction. As no effects on blood pressure or plasma cholesterol were observed, the local effects of ET-1 appear to be independent of these risk factors. This is in line with the most recent study showing that blockade of ET receptors facilitates the maintenance of vasodilation despite high insulin concentrations [37]. In addition, darusentan treatment increased plasma HDL levels in control mice but not in apoE-deficient mice on Western-type diet again suggesting that improvement of endothelial dysfunction in apoE-deficient mice by darusentan treatment was due to the decreased local ET-1 production rather than increased production of plasma HDL. The reason for HDL increase by darusentan in control mice is unknown and remains to be determined.

Enhanced vasoconstriction to serotonin, which is an important factor aggravating ischemia in patients with coronary artery disease, is present in animals and patients with hypercholesterolemia and atherosclerosis [38–41]. In coronary arteries of apoE-deficient mice, fed a high cholesterol diet, was associated with a reduction of endothelium-dependent relaxation to serotonin [30]. We here demonstrate that in addition to impaired endothelium-dependent relaxation in small mesenteric arteries, the contractions to serotonin were enhanced while contractions to norepinephrine remained unaffected in apoE-deficient mice compared to control mice on Western-type diet. This is confirmed by a recent study showing that deletion of eNOS gene in mice is associated with enhanced contraction to serotonin [42]. On the other hand, in isolated human arteries sub-threshold concentrations of ET-1 potentiate contractions to serotonin [43]. Thus, increased tissue ET-1 and endothelial dysfunction in mesenteric arteries may selectively augment serotonin-mediated vasoconstriction. Most interestingly, enhanced vasoconstriction was prevented by chronic ETA receptor blockade, suggesting a role for ETA receptors and/or local ET-1 tissue levels on serotonin-mediated vasoconstriction.

Improvement of vascular function and the reduction of wall thickness after chronic ETA receptor blockade was associated with decreased vascular ET-1 tissue levels comparable to levels found in C57BL/6J control mice. Lerman et al. reported a strong correlation between ET-1 immunoreactivity in the coronary and systemic circulation and the severity of endothelial dysfunction in hypercholesterolemic animals and also in humans [7,8]. The mechanisms by which darusentan normalized elevated ET-1 tissue levels may include ETA-mediated autocrine regulation of ET-1 mRNA [44] or protein synthesis in vivo [4,45] and/or interactions with tissue-bound ET-1 [6]. The beneficial effect of chronic ETA receptor blockade on endothelial function in the vasculature may have important implications for local tissue perfusion and organ function, particularly if atherosclerosis is already present [14,15,46]. The findings suggest that darusentan normalized enhanced vasoreactivity to serotonin and ET-1 and improved endothelium-dependent vasomotion could be clinically relevant by adding novel anti-ischaemic properties to the growth-inhibiting effects of chronic endothelin blockade on structural changes.

Time for primary review 27 days.


   Acknowledgements
 
Supported by the Swiss National Science Foundation (Grants No. 32-51069.97/1 to TFL, No. 32-49648.96 to SS and SCORE 32-58421.99 to MB), the ADUMED Foundation (to LVU and MB), the Novartis Foundation (to LVU) and a grant-in-aid by Knoll AG, Ludwigshafen, Germany. The authors are much indebted to Jane Boden for technical assistance and to Christian Binggeli, M.D. for MATLAB® software programming.


   Notes
 
1 The first two authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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