© 2001 American Heart Association, Inc.
Vascular Biology |
Reactive Oxygen Species Mediate Endothelium-Dependent Relaxations in Tetrahydrobiopterin-Deficient Mice
From Cardiology, Cardiovascular Research (F.C., T.F.L.), University Hospital, Zürich, Switzerland; IRCCS Neuromed (F.C., M.V.), Pozzilli, Italy; Medical Chemistry and Biochemistry (E.R.W.), University of Innsbruck, Innsbruck, Austria; Neurochemistry (J.E.B., M.P.B., S.J.H.), Institute of Neurology, Queen Square, London, UK; Biochemistry (J.R.T.), Imperial College, London, UK; and Cardiovascular Medicine (N.W., K.M.C.), University of Oxford, Oxford, UK.
Correspondence to Thomas F. Lüscher, MD, FHRCP, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. E-mail cardiotfl{at}gmx.ch
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Abstract |
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Abstract—(6R)-5,6,7,8-Tetrahydro-biopterin (H4B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H4B synthesis. A relative shortage of H4B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H4B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BLxCBA) were compared. H4B levels were determined by high-performance liquid chromatography and NO synthase activity by [3H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H4B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with NG-nitro-L-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H4B, the differences between the 2 groups disappeared. Our findings demonstrate that H4B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H4B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.
Key Words: NO synthase • superoxide anion • hydrogen peroxide • catalase GTP cyclohydrolase I
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Introduction |
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(6R)-5,6,7,8-Tetrahydro-biopterin (H4B) is a cofactor of aromatic amino acid hydroxylases, which are regarded as key enzymes in the biosynthesis of several neurotransmitters, including catecholamines and serotonin.1 In addition, H4B is required for the activity of all NO synthase (NOS) isoforms: neuronal NOS, endothelial NOS (eNOS), and inducible NOS.2 3 4 5 6 These enzymes consist of a heme domain linked to an NADPH–cytochrome P-450 reductase–like diflavin domain. On Ca2+/calmodulin binding, the flavin adenine dinucleotide (FAD) transfers reducing equivalents from NADPH to flavin mononucleotide (FMN), which then reduces the heme iron. Reduction of the heme iron leads to oxygen activation followed by oxidation of L-arginine to NO and L-citrulline.7 The precise role of H4B in NO synthesis is not completely understood; however, H4B has been postulated to determine whether the electron flow within the enzyme can be directed to L-arginine.8
NO generated by eNOS contributes to the regulation of systemic blood pressure, and it has been shown to be antiatherogenic and critical for angiogenesis.9 10 Although it is well established that inherent errors in the metabolism of H4B lead to cofactor deficiency, hyperphenylalaninemia, and neurological impairment,11 an important role for H4B in the cardiovascular system has been recognized only recently.12 A close link between cellular H4B availability and NO synthesis has been demonstrated in endothelial cells. Indeed, in porcine and human vascular endothelial cells, inhibition of GTP cyclohydrolase I, the rate-limiting enzyme in H4B synthesis, reduces the production of NO in response to the calcium ionophore A23187 or bradykinin.5 13 14 These studies provided evidence that in cultured endothelial cells, an optimal concentration of H4B is essential for the agonist-induced calcium-dependent production of NO. Furthermore, several biochemical studies have demonstrated that activation of purified constitutive NOS (cNOS) at suboptimal concentrations of the cofactor results in uncoupling of oxygen reduction and arginine oxidation, thereby generating superoxide anion (O2-) and hydrogen peroxide (H2O2).15 16 17 These findings were also confirmed in intact blood vessels depleted of H4B, suggesting that eNOS may become a source of oxygen-derived free radicals.18 19 High-resolution crystal structure of the eNOS heme domain alone and also in complex with H4B has shed light on the role of reduced pterin in sustaining NOS catalysis.20 Under conditions of reduced H4B availability, there is strong evidence for superoxide generation by eNOS.17 This is of physiological interest because H4B depletion is associated with vascular pathology.21
The hypothesis that a relative shortage of H4B may cause a shift in the balance between NOS-catalyzed generation of protective NO and deleterious reactive oxygen species deserves further investigation. If correct, this hypothesis may represent an important mechanism underlying endothelial dysfunction and oxidative vascular injury described in a number of vascular diseases, including atherosclerosis.10 22
Recently, a mouse model of H4B deficiency has been developed by use of the sperm mutagen N-ethyl-N'-nitrosourea.23 The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency in GTP cyclohydrolase I activity, the enzyme catalyzing the first committed step in H4B synthesis.24 This enzyme deficiency results in reduced tissue H4B concentrations compared with concentrations in the wild-type mice of the same strain (C57BLxCBA).25 26
Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H4B deficiency on vascular endothelial function in the intact organism.
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Methods |
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Animals
The experiments were performed on aortas of male hph-1 and wild-type (C57BLxCBA) mice of the same strain (8 weeks old). All procedures were in accordance with institutional guidelines. Body weights of the mice were recorded. Systolic blood pressure and heart rate were measured in conscious mice by a tail-cuff method with the use of a pulse transducer (model LE 5000, Letica). Blood samples were taken in chilled EDTA tubes for the determination of plasma total cholesterol levels. Animals were killed by cervical dislocation. The aortas were excised and immediately placed into cold (4°C) modified Krebs-Ringer bicarbonate solution (control solution containing [mmol/L] NaCl 118.6, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.1, calcium EDTA 0.026, and glucose 10.1). Under a dissection microscope (Wild M3C, Wild AG), the isolated vessels were cleaned of adherent connective tissue. For measurements of H4B levels and eNOS activity, the aortas were frozen in liquid nitrogen and stored at -70°C until assays were performed.
Measurement of Intracellular
Tetrahydrobiopterin
H4B levels were measured in
aortas from both groups of mice. Aortas were homogenized
(5% [wt/vol]) in 0.1 mol/L perchloric acid containing 6.5
mmol/L dithioerythritol, 2.5 mmol/L diethylenetriaminepentaacetic
acid, and 375 U/mL heparin. Samples were centrifuged at
15 000g for 5 minutes, and 100
µL of supernatant was injected onto a high-performance liquid
chromatographic system. Analysis of
H4B was determined as described
previously.27
Determination of cNOS Activity
Determination of cNOS activity was carried out as
described.28 Briefly, tissue
extracts were freed from low molecular weight compounds with NAP-5
columns (Pharmacia). Protein fraction was eluted with 40 mmol/L
Tris-HCl buffer containing 0.1 mg/mL 4-2-(aminoethyl)benzene
sulphonyl fluoride-HCL. Standard reaction mixtures contained 100
mmol/L L-arginine, 25
µmol/L FAD, 25 µmol/L FMN, 2 mmol/L NADPH, 0.15 mmol/L
EGTA, 0.9 mmol/L EDTA, 1.78 mmol/L
MgCl2, 0.27 mmol/L
CaCl2, 5 µmol/L H4B,
and 60 000 to 80 000 cpm purified
L-[2,3,4,5-3H]arginine
(Amersham Life Sciences) and 100 µL of tissue extract in a final
volume of 200 µL. After incubation at 37°C for 30 minutes, the
reaction was stopped by the addition of 800 µL of 200 mmol/L
sodium acetate, pH 5.0, containing 200 µmol/L EDTA and 1 mmol/L
L-citrulline.
[3H]Citrulline was quantified after
separation from [3H]arginine by cation
exchange on Dowex 50W columns.
Measurement of Superoxide by Lucigenin-Mediated
Chemiluminescence
Superoxide
(O2-)
production was determined by a chemiluminescence
method.29 Freshly harvested
aortas were opened lengthwise, divided into multiple segments, and
equilibrated in Krebs-HEPES buffer (composition in mmol/L: NaCl
99, KCl 4.7, MgSO4 1.2,
KH2PO4 1,
CaCl2 1.9, NaHCO3 25,
glucose 11.1, and sodium HEPES 20) gassed with 95%
O2/5% CO2 for 30 minutes
at 37°C in the presence of indomethacin
(10-5 mol/L). Lucigenin-enhanced
chemiluminescence was measured in 2 mL Krebs-HEPES buffer containing
lucigenin (5 µmol/L) by use of a Berthold FB12 single tube
luminometer, modified to maintain a sample temperature of 37°C.
Chemiluminescence was measured continuously for 10 minutes after
allowing dark adaptation and was expressed as relative light units
(RLU) per minute per milligram vessel dry weight.
Organ Chamber Experiments
Aortas were cut into rings (3 to 4 mm long). In
certain rings, the endothelium was mechanically
removed. All experiments were performed in the presence of
indomethacin (10-5 mol/L)
to prevent the formation of prostaglandins. Each ring was
connected to an isometric force transducer (SCAIME), suspended
in an organ chamber filled with 5 mL control solution (37°C, pH 7.4),
and bubbled with 95% O2/5%
CO2. Isometric tension was recorded
continuously. After a 30-minute equilibration period, rings were
gradually stretched to the optimal point of their length-tension curve
(1.5±0.2 g) as determined by the contraction to
norepinephrine (10-6 mol/L).
The functional integrity of the endothelium was tested
by the presence of relaxations to acetylcholine
(10-6 mol/L). Concentration-response
curves were obtained in a cumulative fashion. Several rings cut from
the same artery were studied in parallel; only 1 concentration-response
curve was made per preparation. In quiescent preparations,
indomethacin,
NG-nitro-L-arginine
methyl ester (L-NAME), catalase, and superoxide dismutase (SOD) did not
affect resting tension. Responses to acetylcholine were obtained during
submaximal contraction to norepinephrine
(10-6 mol/L). Incubation time was 60
minutes for H4B, 30 minutes for
indomethacin, 15 minutes for L-NAME, and 5 minutes for
catalase and SOD. Relaxations were expressed as a percentage of maximal
relaxations induced by papaverine (3x10-4
mol/L).
Drugs
Acetylcholine chloride, catalase (C-100 from bovine
liver, 58 000 U/mg protein), indomethacin, L-NAME,
norepinephrine, papaverine hydrochloride, sodium
nitroprusside, SOD (from bovine erythrocytes, 4400 U/mg protein), and
chemical components of the control solution were obtained from Sigma
Chemical Company. H4B dihydrochloride was
obtained from Schircks Laboratories and prepared just before
administration with the use of oxygen-free distilled water. Stock
solutions of the drugs were freshly prepared every day. A stock
solution of 10-5 mol/L
indomethacin was prepared in equimolar concentrations
of Na2CO3. All
concentrations are expressed as final molar concentration in the bath
solutions.
Statistical Analysis
All experiments were performed in parallel on
preparations from hph-1 and wild-type mice. In all experiments, n
equals the number of mice per experiment. Results are expressed as
mean±SEM. Statistical evaluation of the data was performed by using
Student t test for simple
comparison between 2 values when appropriate. For multiple comparisons,
results were analyzed by ANOVA. A value of
P<0.05 was considered
statistically significant.
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Results |
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Characteristics of Animals
The body weight, systolic blood pressure, heart rate, and plasma total cholesterol levels of 8-week-old male hph-1 and wild-type (C57BLxCBA) mice of the same strain are shown in Table 1
. In hph-1 mice, the systolic blood pressure
was higher than that in the wild-type control
mice.
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Levels of H4B and
Measurements of cNOS Activity
The intracellular levels of H4B
in the aortas obtained from both groups were significantly lower in the
mutant compared with the wild-type control mice
(Figure 1A). Accordingly, cNOS activity assayed without the
addition of exogenous H4B was significantly
lower in hph-1 aortas (0.8±0.03 and 1.9±0.5
pmol · mg-1 · minute-1,
respectively; n=3; P<0.05;
Figure 1B). By contrast, cNOS activity assayed with the
addition of exogenous H4B
(10-5 mol/L) was comparable between the 2
groups (10±0.7 and 7.4±0.9
pmol · mg-1 ·
minute-1, respectively;
Figure 1B).
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Superoxide Anion Generation
O2-
production was measured in the aortas of wild-type and hph-1
mice under basal conditions and after inhibition of NOS
(Figure 2). Basal
O2- generation from
hph-1 was slightly elevated compared with that from wild-type controls
(58±6 versus 40±1
RLU · s-1 · mg-1,
respectively; n=9). Furthermore, in mutant mice,
O2- release was
significantly decreased after incubation with
NG-monomethyl-L-arginine
(L-NMMA, 10-3 mol/L; 26±3 versus 58±6
RLU · s-1 ·
mg-1, respectively; n=9;
P<0.05). By contrast,
preincubation of control mouse aortas with L-NMMA resulted in an
increase in O2-
release (107±10 versus 40±1
RLU · s-1 · mg-1,
respectively; n=9;
P<0.05)
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Vascular Contractions to
Norepinephrine
The contractions to norepinephrine
(10-6 mol/L) did not differ between the 2
groups
(Table 2). Furthermore, the treatments with L-NAME,
catalase, superoxide dismutase, or exogenous H4B
did not affect the response to norepinephrine
(Table 2).
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Endothelium-Dependent
Relaxations to Acetylcholine
During contractions to norepinephrine
(10-6 mol/L), acetylcholine
(10-9 to 10-6
mol/L) caused endothelium-dependent relaxations in
mutant and wild-type mice
(Figure 3). Inhibition of NOS with L-NAME
(3x10-4 mol/L) abolished the relaxations
to acetylcholine in both groups
(Figure 3, P<0.05
versus control). However, only in mutant mice were the relaxations
significantly inhibited by catalase (1200 U/mL,
Figure 3, P<0.05
versus control). The inhibitory effect of catalase
disappeared after incubation of mutant mouse aortas with exogenous
H4B (10-4 mol/L,
Figure 4). Furthermore, SOD (150 U/mL) enhanced the
acetylcholine-induced relaxations only in hph-1 mice
(Figure 5, P<0.05
versus control).
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Endothelium-Indepedent
Relaxations to Sodium Nitroprusside
During contraction induced with
norepinephrine, the NO donor sodium nitroprusside
(10-10 to
10-5 mol/L) caused similar
concentration-dependent relaxations in both groups. Catalase did not
affect the relaxations to sodium nitroprusside
(Figure 6).
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Discussion |
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The present study demonstrates that in an animal model of H4B deficiency, endothelium-dependent relaxations are in part mediated by eNOS-catalyzed production of oxygen-derived free radicals. This conclusion is supported by several lines of evidence. In spite of lower intracellular levels of H4B and concomitant loss of cNOS activity in hph-1 compared with wild-type mice, the endothelium-dependent relaxations to acetylcholine were present in the aortas of both groups. Inhibition of NOS with L-NAME abolished these relaxations in H4B-deficient mice as well as in control mice. Only in mutant mice were the endothelium-dependent relaxations inhibited by catalase, a selective scavenger of H2O2. In the presence of exogenous H4B, the inhibitory effect of catalase disappeared. Furthermore, relaxations to acetylcholine in hph-1 mice were enhanced by SOD, an enzyme that rapidly converts O2- into H2O2 and oxygen. Accordingly, O2- production was significantly decreased in hph-1 mice after NOS inhibition. These findings suggest that under conditions of in vivo H4B deficiency, H2O2, a product of O2- dismutation formed via eNOS, becomes a mediator of endothelium-dependent relaxations.
It is well established that all NOS isoforms catalyze a 5-electron oxidation of L-arginine to L-citrulline, producing stoichiometric amounts of NO in the process. Therefore, our observation of a reduced cNOS activity (measured as production of L-citrulline) that is associated with apparently normal relaxations to acetylcholine in hph-1 mice is consistent with the concept of a dysfunctional NOS. A relative shortage of H4B may indeed lead to an uncoupling between L-arginine oxidation and production of L-citrulline and NO, resulting in a shift between NOS-dependent generation of NO and reactive oxygen species with vasorelaxing properties. Previous studies30 31 have provided evidence that H2O2 is a potent vasodilator through direct activation of soluble guanylate cyclase in smooth muscle cells and an increase in cGMP. The present findings are in agreement with previously reported results in which a dysfunction of eNOS occurred in isolated arteries and cultured endothelial cells depleted of H4B.5 13 18 19 In canine coronary arteries incubated with the GTP cyclohydrolase I inhibitor 2,4-diamino-6-hydroxypyrimidine, endothelium-dependent relaxations to calcium ionophore and its stimulating effect on cGMP production were reduced by the H2O2 scavenger catalase.18 The present study clearly strengthens the concept of an interrelationship between the availability of H4B and eNOS activity. NOS-catalyzed conversion of L-arginine to L-citrulline and NO exhibits unique complexity and requirements for cofactors.6 In the presence of optimal concentrations of H4B, L-arginine undergoes oxidative cleavage to yield NO and L-citrulline. The required reducing equivalents are derived from NADPH and are shuttled through the reduced flavins, FAD and FMN to the heme group.32 33 34 This built-in electron transport system is used to oxidize L-arginine to NO and L-citrulline. H4B fully couples L-arginine oxidation to NADPH consumption. Indeed, in the presence of suboptimal levels of H4B, there is strong evidence for O2- and H2O2 generation by purified eNOS.17 20 Furthermore, it was recently found that increased generation of O2- in cultured endothelial cells exposed to LDLs can be inhibited by L-NAME.35 These data support the hypothesis that NOS itself can be an important source for the endothelial production of reactive oxygen species. In our present study, confirmation that NOS contributes to O2- generation in hph-1 mice comes from experiments showing a significant inhibition of O2- release after incubation with L-NMMA. By contrast, preincubation of control mouse aortas with the L-arginine analogue resulted in an increase in vascular O2- release. It is likely that in wild-type aortas, the activity of common sources of O2-, such as NADPH oxidase, cyclooxygenase, and xanthine-oxidase associated with the inhibition of NOS-catalyzed generation of NO, results in the loss of the NO-scavenging effect and, hence, increased O2- concentration. The opposite effect exerted by L-NMMA in mutant mice suggests that NO production is already impaired in these animals and that NOS catalytic activity is shifted toward O2- generation. Because all the experiments in the present study were performed in the presence of indomethacin, it is possible to rule out the possibility that oxygen-derived free radical production was initiated by the activation of arachidonic acid metabolism via the cyclooxygenase pathway. Also, we ruled out the possibility that reactive oxygen species are produced by smooth muscle cells in hph-1 aortas. Indeed, catalase did not affect relaxations to the NO donor sodium nitroprusside in preparations without endothelium.
To validate the decreased intracellular availability of H4B as the mechanism leading to such eNOS-catalyzed production of oxygen-derived free radicals, we measured the levels of H4B and the activity of cNOS in the vessel wall. Under basal conditions, these measurements revealed a significantly lower tissue concentration of the cofactor as well as loss of NOS activity (measured as picomoles of L-citrulline produced per milligram of aortic tissue per minute) in aortas from mutant mice compared with those from wild-type control mice. However, with the addition of exogenous H4B, the activity of cNOS was comparable between the 2 groups. The same findings were obtained for brain concentrations of H4B and whole-brain NOS activity in the hph-1 mice.26 In spite of such reduced enzymatic activity, relaxations to acetylcholine were similar to those in the control group and were abolished by the inhibition of NOS with L-NAME as well. However, the selective effects of catalase and SOD on the acetylcholine-induced responses only in H4B-deficient mice clearly indicate that the endothelium-dependent relaxations are in part mediated by eNOS-catalyzed generation of reactive oxygen species. Accordingly, incubation of hph-1 aortas with supplementary H4B abolished the inhibitory effect of catalase on endothelium-dependent relaxations to acetylcholine. This shows that an optimal concentration of H4B is critical for calcium-dependent production of NO and L-citrulline.
An important aspect of the present study is that our findings for the first time were obtained in an in vivo model of chronic deficiency of H4B and not through a pharmacologically induced depletion of the cofactor.
Although the endothelium-dependent relaxations to acetylcholine are apparently unchanged in H4B-deficient mice compared with wild-type control mice, an increased generation of O2- and its dismutase product H2O2 in the long-term may contribute to endothelial dysfunction. Our results do not allow any conclusion regarding the relative amount of reactive oxygen species versus NO produced, but such NOS-catalyzed formation of O2- and its subsequent dismutation into H2O2 and OH radical or its transformation into peroxynitrite cleavage products may represent an important mechanism underlying oxidative injury described in a number of vascular diseases.36 Indeed, recent clinical studies have demonstrated that administration of H4B improves endothelial dysfunction in conditions characterized by reduced availability of NO and increased production of reactive oxygen species, such as hypercholesterolemia,37 coronary artery disease,38 and smoking.39 40 41 Several other reports suggest that H4B may play a role in endothelial dysfunction during hypertension,21 ischemia/reperfusion,42 and experimental diabetes.43 In this regard, hph-1 mice had a higher systolic blood pressure compared with that in control mice. It is of interest to note that basal NOS activity was reduced in mutant mice. Blockade of basal NO production by specific inhibitors of NOS induces persistent hypertension in animals44 45 and increases blood pressure in humans.46 Furthermore, basal NOS activity is reduced in hypertensive patients, as judged by the urinary [15N]nitrate excretion.47 Thus, our findings suggest the importance of eNOS dysfunction in the setting of reduced H4B levels for blood pressure regulation. In line with this interpretation, we have previously found that an impaired synthesis of H4B in prehypertensive rats may contribute to the development of hypertension or its complications.21 However, a variety of other mechanisms, such as neurological disorders, microcirculatory rarefaction, and renal aspects, might account for hypertension in hph-1 mice. There is substantial evidence to rule out an increased activity of the autonomic nervous system. Indeed, this in vivo model of H4B deficiency is characterized by an impaired biosynthesis of catecholamines and serotonin.48
Taken together, all these data suggest that conditions associated with impaired NO activity and accelerated atherosclerosis may be characterized by a reduced availability of H4B. The background for such a reduced availability is not clear.49 Decreases in cellular H4B concentration and in H4B binding affinity may alter eNOS activity in favor of oxygen-derived free radical generation.21 The identification of signal transduction pathways involved in the control of gene expression and activity of GTP cyclohydrolase I50 as well as characterization of the precise role of H4B in regulation of NOS catalytic activity51 20 will help us to understand how an impairment of H4B availability may take place in vascular diseases and provide new targets for pharmacotherapy.
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Acknowledgments |
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This work was supported in part by grants from the Swiss National Research Foundation (No. 32-510069.97), the Italian National Research Council Project (No. 99.00171.PF34), the Research Trust for Metabolic Disease in Children (UK), and the Austrian Research Funds Project (No. 13793). We would like to thank Sandro Bonetti and Dr Laura Canevari for blood pressure and plasma cholesterol measurements, respectively. We would also like to thank Petra Hoefler for technical assistance.
Received September 29, 2000; accepted December 20, 2000.
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