Journal of the American College of Cardiology
© 2008 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 52, No. 2, 2008
ISSN 0735-1097/08/$34.00
doi:10.1016/j.jacc.2007.12.062
Vascular Disease
GCH1 Haplotype Determines Vascular and
Plasma Biopterin Availability in Coronary Artery Disease
Effects on Vascular Superoxide Production and Endothelial Function
Charalambos Antoniades, MD, PHD,* Cheerag Shirodaria, MRCP,* Tim Van Assche, PHD,*
Colin Cunnington, MRCP,* Irmgard Tegeder, MD, PHD,†‡ Jörn Lötsch, MD, PHD,‡
Tomasz J. Guzik, MD, PHD,*§ Paul Leeson, MRCP, PHD,* Jonathan Diesch, HNC,*
Dimitris Tousoulis, MD, PHD, FACC,¶ Christodoulos Stefanadis, MD, FACC, FESC,¶
Michael Costigan, PHD, Clifford J. Woolf, MD, PHD,† Nicholas J. Alp, MD, PHD, MRCP,*
Keith M. Channon, MD, FRCP*
Oxford, United Kingdom; Boston, Massachusetts; Frankfurt am Main, Germany; Cracow, Poland; and Athens, Greece
Objectives
This study sought to determine the effects of endogenous tetrahydrobiopterin (BH4) bioavailability on endothelial nitric oxide synthase (eNOS) coupling, nitric oxide (NO) bioavailability, and vascular superoxide production in
patients with coronary artery disease (CAD).
Background
GTP-cyclohydrolase I, encoded by the GCH1 gene, is the rate-limiting enzyme in the biosynthesis of BH4, an
eNOS cofactor important for maintaining enzymatic coupling. We examined the associations between haplotypes of the GCH1 gene, GCH1 expression and biopterin levels, and the effects on endothelial function and vascular superoxide production.
Methods
Blood samples and segments of internal mammary arteries and saphenous veins were obtained from patients with
CAD undergoing coronary artery bypass grafting (n ⫽ 347). The GCH1 haplotypes were defined by 3 polymorphisms:
rs8007267G⬍A, rs3783641A⬍T, and rs10483639C⬍G (X haplotype: A, T, G; O haplotype: any other combination).
Vascular superoxide (⫾ the eNOS inhibitor NG-nitro-L-arginine methyl ester [L-NAME]) was measured by lucigeninenhanced chemiluminescence, whereas the vasorelaxations of saphenous veins to acetylcholine were evaluated ex vivo.
Results
Haplotype frequencies were OO 70.6%, XO 27.4%, and XX 2.0%. The X haplotype was associated with significantly
lower vascular GCH1 messenger ribonucleic acid expression and substantial reductions in both plasma and vascular BH4 levels. In X haplotype carriers both vascular superoxide and L-NAME–inhibitable superoxide were significantly increased, and were associated with reduced vasorelaxations to acetylcholine.
Conclusions
GCH1 gene expression, modulated by a particular GCH1 haplotype, is a major determinant of BH4 bioavailability both
in plasma and in the vascular wall in patients with CAD. Genetic variation in GCH1 underlies important differences in
endogenous BH4 availability and is a determinant of eNOS coupling, vascular redox state, and endothelial function in
human vascular disease. (J Am Coll Cardiol 2008;52:158–65) © 2008 by the American College of Cardiology Foundation
Maintenance of endothelial function is a critical aspect of
vascular homeostasis. Loss of normal endothelial production
of nitric oxide (NO) is an early and characteristic feature of
vascular disease states and plays a role in disease pathogenesis (1). Endothelial nitric oxide synthase (eNOS) is reguSee page 166
From the *Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; †Neural Plasticity Research Group,
Department of Anesthesia and Critical Care, Massachusetts General Hospital and
Harvard Medical School, Boston, Massachusetts; ‡Pharmazentrum Frankfurt/ZAFES,
Institute for Clinical Pharmacology, Johann Wolfgang Goethe University, Frankfurt am
Main, Germany; §Departments of Medicine and Pharmacology, Jagiellonian University,
Cracow, Poland; and the ¶Athens University Medical School, 1st Cardiology Department, Hippokration Hospital, Athens, Greece. This study was supported by the Marie
Curie Intra-European Fellowship, within the 6th European Community Framework
Programme (Dr. Antoniades). This work was also supported by the National Institute
of Health Research Oxford Biomedical Research Centre Programme, and by grants
from the British Heart Foundation (RG/02/006 to Dr. Channon, FS/03/105/16340 to Dr.
Shirodaria). Drs. Costigan and Woolf were supported by the National Institutes of Health.
Manuscript received November 2, 2007; accepted December 12, 2007.
lated by the cofactor tetrahydrobiopterin (BH4) (2). Reduced BH4 availability in disease states seems to be an
important aspect of impaired eNOS activity and increased
vascular superoxide production (1,2). In humans with vascular disease states, acute administration of BH4 can improve endothelial function (3,4), and treatments such as
folates or vitamin C, which improve endothelial function,
may exert some of their effects by increasing BH4 availability (5–9). However, the extent to which alterations in endogenous BH4 availability, rather than pharmacologic effects of
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
BH4, might play a direct role in modulating endothelial
function in humans in vivo remains unclear.
GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of BH4 (10), and is a major
determinant of BH4 levels, through transcriptional regulation
of GCH1 expression. In some experimental models of vascular
disease, reduced BH4 is associated with reduced GTPCH
protein levels, suggesting reduced BH4 biosynthesis (11).
However, BH4 oxidation by free radicals such as peroxynitrite
(ONOO⫺), generating dihydrobiopterin (BH2) and biopterin
(B), may cause loss of BH4 without changes in biopterin
synthesis (5,7,12). The relative importance of synthesis versus
oxidation of BH4 in atherosclerosis is complex because both
local and systemic inflammation up-regulate GCH1 expression
(13) but also increase ONOO⫺ production, potentially resulting in greater BH4 oxidation (14).
In genetic mouse models, constitutive reduction in GCH1
expression causes BH4 deficiency, resulting in abnormal
eNOS regulation (14 –16). Conversely, transgenic overexpression of GTPCH in the vascular endothelium is sufficient to augment vascular BH4 levels (17), improve endothelial function in models of vascular disease (14), and
rescue the vascular effects of genetic BH4 deficiency (16).
Thus, in experimental models, genetic alteration of endogenous BH4 biosynthesis has provided useful insights into
the role of BH4 in vascular disease pathogenesis. In contrast, it is unknown whether alterations in endogenous BH4
synthesis in humans are sufficient to alter systemic or
vascular BH4 availability, or have any impact on vascular
function.
We have recently identified a novel haplotype of the
GCH1 gene that is associated with significantly lower
GTPCH activity in stimulated leukocytes (18,19). This
haplotype is defined by 3 single nucleotide polymorphisms: rs8007267G⬎A in the putative promoter region,
rs3783641A⬎T in intron 1 and rs10483639C⬎G in the 3=
untranslated region of the GCH1 gene (18,19).
In this study we examined the functional effects of this
GCH1 haplotype on the levels of BH4 and total biopterins
both in plasma and in vessels obtained from patients with
coronary artery disease (CAD). Furthermore, we investigated how intrinsic genetic variation in vascular BH4
biosynthesis alters regulation of eNOS function in the
human vasculature through effects on superoxide production
and NO-mediated endothelial function.
Methods
Study subjects. We studied 347 patients with CAD undergoing elective coronary artery bypass grafting (CABG) at
the John Radcliffe Hospital, Oxford, United Kingdom.
Exclusion criteria were any inflammatory, infective, liver, or
renal disease; malignancy; acute coronary event during the
last 2 months; or clinically overt heart failure. Patients
receiving nonsteroidal anti-inflammatory drugs, dietary supplements of folic acid, or antioxidant vitamins were also
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
159
excluded. Demographic characAbbreviations
teristics of the patients are preand Acronyms
sented in Table 1. The study was
ACh ⴝ acetylcholine
approved by the local research
BH4 ⴝ tetrahydrobiopterin
ethics committee, and each patient
BH2 ⴝ dihydrobiopterin
gave written informed consent.
B ⴝ biopterin
Plasma and tissue samples.
CABG
ⴝ coronary artery
Blood samples were obtained imbypass
grafting
mediately before surgery, after
CAD
ⴝ
coronary artery
overnight fasting. Samples were
disease
centrifuged at 2,500 rpm for 10
CRP ⴝ C-reactive protein
min, and serum or plasma was
o
eNOS ⴝ endothelial nitric
stored at ⫺80 C until assayed.
oxide synthase
Samples of saphenous vein (SV)
GTPCH ⴝ GTPand internal mammary artery
cyclohydrolase I
(IMA) were obtained at the time
IMA ⴝ internal mammary
of CABG surgery, as we have
artery
previously described (5,20). Paired
L-NAME ⴝ NG-nitro-Lvessel segments were snap-frozen
arginine methyl ester
o
and stored at ⫺80 C for meaNO ⴝ nitric oxide
surement of biopterin content, or
O2ⴚ ⴝ superoxide radical
were transferred to the laboratory
ONOOⴚ ⴝ peroxynitrite
for functional studies within 30
Ox-LDL ⴝ oxidized lowmin, in ice cold Krebs-Henseleit
density
lipoprotein
buffer (5,20).
RT-qPCR
ⴝ real-time
Deoxyribonucleic acid (DNA)
quantitative polymerase
extraction and genotyping.
chain reaction
Genomic DNA was extracted
SNP ⴝ sodium
from 2 ml of whole blood using
nitroprusside
standard methods (QIAamp
SV ⴝ saphenous vein.
DNA blood Midi kit, Qiagen,
Germantown, Maryland). In addition, DNA samples were available from 741 unrelated
subjects of Caucasian ethnicity (mostly medical students)
who had consented into genotyping and served as control
subjects for allelic frequencies in the present study.
The DNA samples were screened for that particular
functional GCH1 haplotype identified as being associated
with decreased GCH1 expression and BH4 production in a
previous study (21). The complete haplotype consists of 15
DNA positions of the GCH1 gene. However, as described
elsewhere (19), the diagnosis of the particular GCH1 haplotype of present interest is possible with 100% sensitivity
and specificity by screening for just 3 GCH1 singlenucleotide polymorphisms that span the entire DNA range
of the haplotype: c. –9610G⬎A (dbSNP rs8007267G⬎A)
in the 5= untranslated region, c.343⫹8900A⬎T (dbSNP
rs3783641A⬎T) in intron 1, and c.*4279 (dbSNP
rs10483639C⬎G) in the 3= untranslated region. Thus, for
diagnosis purposes the haplotype of interest reduces to
rs8007267A/rs3783641T/rs10483639G. The GCH1 haplotype of interest was identified by means of validated Pyrosequencing screening assays (Pyrosequencer PSQ95, Uppsala,
Sweden) diagnosing the 3 selected sodium nitroprussides
(SNPs) (19). Two different types of positive control samples were implemented into all Pyrosequencing screening
160
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
Demographic Characteristics of Patients
Table 1
Demographic Characteristics of Patients
Number of patients
Male/female
Age (yrs)
347
292/55
65.42 ⫾ 0.49
Saphenous veins (n)
289
Internal mammary arteries (n)
166
Risk factors
Hypertension (%)
72.7
Hypercholesterolemia (%)
71.6
Smokers (current/ex) (%)
8.6/69.8
Diabetes mellitus (%)
27.5
Family history for coronary artery disease (%)
63.3
Body mass index (kg/m2)
Triglycerides (mmol/l)
28.04 ⫾ 0.34
1.65 ⫾ 0.09
Cholesterol (mmol/l)
4.07 ⫾ 0.08
High-density lipoprotein (mmol/l)
1.10 ⫾ 0.02
C-reactive protein (mg/l)
2.48 ⫾ 0.70
Plasma biopterin levels
Plasma tetrahydrobiopterin (nmol/l)
20.46 (10.13–41.86)
Plasma dihydrobiopterin (nmol/l)
15.19 (11.77–19.41)
Plasma biopterin (nmol/l)
Plasma total biopterins (nmol/l)
BH4/tBio ratio
4.09 (2.83–6.98)
46.11 (30.04–71.33)
0.49 ⫾ 0.018
Medication (%)
Statins
90.0
ACEI/ARB
66.0
Calcium-channel blockers
40.8
Beta-blocker
74.6
Aspirin
87.1
Values expressed as mean ⫾ SEM or median (25th to 75th percentile) unless otherwise noted.
ACEI ⫽ angiotensin-converting enzyme inhibitors; ARB ⫽ angiotensin receptor blockers; BH4 ⫽
tetrahydrobiopterin; tBio ⫽ total biopterins.
analyses. One positive control set contained DNA samples from carriers of the haplotype independently diagnosed
for all 15 GCH1 DNA positions by the 5=-exonuclease
method (22). The second set of positive controls consisted
of DNA samples homozygous or heterozygous for the
frequent or rare alleles at the 3 SNP positions at the GCH1
DNA, diagnosed externally by means of conventional sequencing (AGOWA GmbH, Berlin, Germany) (18,19).
Ribonucleic acid (RNA) isolation and real-time quantitative polymerase chain reaction (RT qPCR). Snapfrozen vascular rings (IMA and SV) were initially lysed in
Trizol reagent (Tri-Reagent, Sigma, St. Louis, Missouri),
followed by RNA purification from the aqueous phase using
the RNeasy Micro kit (Qiagen, Stanford, California). Ribonucleic acid was converted into complementary DNA
(Superscript II reverse transcriptase, Invitrogen, Carlsbad,
California), then subjected to qPCR using the TaqMan
system (Applied Biosystems, Foster City, California; Assay
ID GCH ⫽ Hs00609198_m1, Assay ID GAPDH ⫽
Hs02758991_g1) and analyzed on an iCyclerIQ (Biorad,
Hercules, California). Relative expression was calculated using
the 2⫺⌬⌬CT method.
Determination of plasma and vascular biopterin levels. BH4,
BH2, and biopterin levels in plasma or vessel tissue lysates
were each determined separately from the same sample, by
high-performance liquid chromatography followed by serial
electrochemical and fluorescent detection, as previously
described (23). Total biopterins were quantified by summing BH4, BH2, and B. Biopterin levels were expressed as
pmol/g of tissue for vessels and nmol/l for plasma.
Determination of vascular superoxide production. Vascular superoxide production was measured in paired segments of SV using lucigenin-enhanced chemiluminescence
as previously described (5,24). Vessels were opened longitudinally to expose the endothelial surface and equilibrated
for 20 min in oxygenated (95% O2/5% CO2) Krebs-4-(2hydroxyethyl)-1-piperazine-ethane-sulfonic acid buffer (pH
⫽ 7.4) at 37°C. Lucigenin-enhanced chemiluminescence
was measured using low-concentration lucigenin (5 ␮mol/l)
(25) because higher concentrations of lucigenin (up to 250
␮mol/l) favor redox cycling (24). As a measure of eNOS
coupling we determined NOS-derived superoxide production, which was estimated as the difference in superoxide
production after 20 min incubation with the NOS inhibitor
N G -nitro-L-arginine methyl ester (L-NAME) (100
␮mol/l).
Vasomotor studies. Endothelium-dependent and -independent
dilatations were assessed in SV obtained at the time of CABG,
using isometric tension studies (5,26). Four rings from each
vessel were pre-contracted with phenylephrine (3 ⫻ 10⫺6
␮mol/l), then endothelium-dependent relaxations were quantified using acetylcholine (ACh, 10⫺9 to 10⫺5 ␮mol/l).
Finally, relaxations to the endothelium-independent NO
donor SNP (10⫺10 to 10⫺6 ␮mol/l) were evaluated in the
presence of L-NAME (100 ␮mol/l) as we have previously
described (5,26).
Determination of oxidized low-density lipoprotein (oxLDL) and C-reactive protein (CRP) levels. Serum levels
of ox-LDL were measured by enzyme-linked immunosorbent assay using commercially available kit (Mercodia,
Sweden). Serum levels of CRP were measured by immunonephelometry using a high-sensitivity method (Dade
Behring Marburg GmbH, Marburg, Germany).
Statistical analysis. GCH1 haploblocks and linkage disequilibrium among the 3 SNPs with parameters D= and r2
(27,28) were analyzed using the solid spine of LD algorithm
implemented in Haploview (29). Allelic frequencies of the
patient cohorts were compared with those observed in a
random sample of 741 healthy unrelated control subjects of
Caucasian ethnicity (mainly medical students of the University of Frankfurt am Main, Germany) by means of the
Fisher exact test. On the basis of the observed allelic
frequency, the expected number of homozygous and heterozygous carriers of the respective SNP was calculated
using the Hardy-Weinberg equation as p2 ⫹ 2pq ⫹ q2 ⫽ 1,
where p and q are defined as the probabilities of occurrence
for the dominant and mutated alleles, respectively (30). The
correspondence between the observed numbers of homozygous and heterozygous individuals and the numbers expected on the basis of the Hardy-Weinberg equilibrium,
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
150
Plasma tBio (nmol/L)
Plasma BH4 (nmol/L)
B
100
P<0.01
50
0
C 50
P<0.01
100
50
0
D 200
SV tBio (pmol/g)
P<0.01
40
P<0.01
150
30
100
20
10
E
50
0
0
60
F 300
P<0.01
IMA tBio (pmol/g)
We defined GCH1 haplotypes in 347 patients with CAD
and 741 control subjects. In CAD patients, the 3 SNPs used
to define the haplotypes were in linkage disequilibrium.
Specifically, values of D= and r2 were 95 and 77 for
rs8007267 and rs3783641, 87 and 69 for rs3783641 and
rs10483639, and 86 and 5 for rs8007267 and rs10483639,
respectively. The 3 SNPs were located within 1 single
haploblock (29). The numbers of 245 noncarriers (OO) and
95 heterozygous (XO) and 7 homozygous (XX) carriers of
the GCH1 X haplotype was in accordance with the HardyWeinberg law (chi-square goodness of fit test: p ⫽ 0.81).
Hardy-Weinberg equilibrium also applied to the control
subjects (OO, n ⫽ 543; XO, n ⫽ 178; XX, n ⫽ 20;
chi-square goodness of fit test: p ⫽ 0.51). The allelic
frequencies of the GCH1 X haplotype of 15.7 and 14.7 in
patients and healthy control subjects, respectively, did not
A
SV BH4 (pmol/g)
Results
differ between groups (Fisher exact test: p ⫽ 0.56). Moreover, the numbers of noncarriers and heterozygous and
homozygous carriers of the GCH1 X haplotype were similar
in patients and control subjects (chi-square test: p ⫽ 0.42).
We first examined the effect of GCH1 haplotype on the
levels of plasma biopterins. Patients with the X haplotype
had significantly lower plasma levels of both BH4 and total
biopterins than patients homozygous for the common O
haplotype; median plasma BH4 levels in patients with XX
genotype were reduced by approximately 80% compared
with OO patients. Indeed, plasma BH4 and total biopterin
levels were reduced in a striking allele-dependent manner
from OO, XO, and XX genotypes (Fig. 1). Because vascular
biopterin levels are likely more important for eNOS
regulation, and could plausibly be differentially regulated
from plasma biopterin levels, we next sought to examine
the effect of GCH1 haplotype on vascular biopterins, in
both SV and IMA. The presence of the X haplotype was
associated with significantly lower levels of vascular BH4
and total biopterins in both SV and IMA (Fig. 1). These
findings suggest a direct effect of this GCH1 haplotype on
biopterin synthesis.
IMA BH4 (pmol/g)
indicating that the study sample corresponded to a random
sample of subjects, was assessed using the chi-square goodness of fit test. Allelic frequencies of the cohort of 347
patients were compared with those observed in a random
sample of 741 healthy unrelated control subjects of Caucasian ethnicity (mainly medical students of the University of
Frankfurt am Main, Germany) by means of the Fisher exact
test. Between these cohorts, the number of noncarriers,
homozygous carriers, and heterozygous carriers of the particular GCH1 haplotype were compared using chi-square
statistics.
All variables were tested for normal distribution using the
Kolmokorov-Smirnov test. Normally distributed variables
are presented as mean ⫾ SEM, whereas nonnormally
distributed variables were log-transformed for analysis and
are presented as median [25th to 75th percentiles] and
range. Comparisons of baseline and demographic characteristics among the 3 genotypes were performed using
one-way analysis of variance for multiple comparisons,
whereas an unpaired t test was used to compare variables
between 2 groups (for recessive models).
Univariate analysis was performed by calculation of the
Pearson coefficient. Multivariate analysis was used to examine the effect of the genotype on vascular/plasma biopterins,
vascular superoxide, L-NAME–induced change of vascular
superoxide, or maximum relaxations to ACh as dependent
variables. As independent variables we used GCH1 genotype, and those clinical characteristics (age, gender, diabetes
mellitus, smoking, dyslipidemia, hypertension, body mass
index, and medications) that showed an association with the
dependent variable in univariate analysis at the level of 15%.
A backward elimination procedure was applied in all models, having p ⬎ 0.1 as the threshold to remove a variable
from the model. All p values were 2-tailed, and a value of
p ⬍ 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 12.0
(SPSS Inc., Chicago, Illinois).
161
40
20
0
200
100
0
OO
Figure 1
P<0.05
XO
XX
OO
XO
XX
Effect of GCH1 Haplotype on Plasma/Vascular BH4
The presence of the haplotype (XO or XX) was associated with significantly
lower levels of plasma tetrahydrobiopterin (BH4) (A) and total biopterins (tBio)
(B). A similar effect was also observed in saphenous veins (SV) (C and D) and
internal mammary arteries (IMA) (E and F).
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
Figure 2
Effect of GCH1 Haplotype
on Vascular GTPCH mRNA Levels
The presence of the XX genotype was associated with significantly lower messenger ribonucleic acid (mRNA) levels of GTPCH compared with OO genotype.
Shown are the data derived from 23 saphenous veins (SVs) and 17 internal
mammary arteries (IMAs). *p ⬍ 0.05 versus OO.
B
4
P<0.01
3
2
1
LNAME Δ[O2-]
(RLU/sec/mg)
Vascular O2(RLU/sec/mg)
A
0
-1
-2
-3
P<0.01
0
0.4
0.2
0
E
30
56
P<0.05
52
48
44
40
P<0.05
20
10
0
OO XO+XX
Figure 3
D
Oxidized-LDL (IU/L)
P<0.05
0.6
F
SNP relaxation (%)
C
BH4:tBio Ratio
To investigate whether the effect of the GCH1 haplotype on
biopterin levels was mediated by changes in GCH1 gene
expression, we performed RT-PCR to quantify GCH1 messenger (m)RNA in samples of SV and IMA from patients with
different GCH1 genotypes. We observed that the XX genotype
was associated with significantly reduced vascular GCH1 expression compared with the OO genotype (Fig. 2). Furthermore, vascular GCH1 mRNA levels across all genotypes were
significantly correlated with both plasma (r ⫽ 0.394, p ⫽
0.010) and vascular (r ⫽ 0.336, p ⫽ 0.042) BH4 levels.
We next sought to investigate how these genetically determined differences in biopterin levels might influence NOmediated endothelial function and vascular superoxide production. Because homozygosity for the X haplotype is relatively
uncommon (2% of our population), we used a recessive model,
comparing patients with or without X haplotype. In support of
the recessive model, patients with at least 1 X haplotype (i.e.,
XO or XX genotypes) had significantly lower BH4 and total
biopterins than OO patients, in both plasma (16.6 [5.2 to 28.3]
and 41.2 [23.4 to 54.9] nmol/l vs. 21.57 [12.1 to 45.24] and
48.3 [32.6 to 76.4] nmol/l, p ⬍ 0.05 for both) and SV (7.1 [2.2
to 9.3] and 32 [22 to 93] nmol/l vs. 11.0 [5.7 to 17.8] and 43
[24 to 76] nmol/g, p ⬍ 0.05 for both).
Vascular superoxide production was significantly increased in X haplotype carriers (Fig. 3). To explore the
specific contribution of eNOS coupling, we quantified the
difference in vascular superoxide production after NOS
inhibition by L-NAME. Incubation of vessels with
L-NAME inhibited superoxide production to a significantly greater extent in X haplotype carriers than in OO
homozygotes (Fig. 3). Increased oxidative stress in X haplotype carriers was also supported by the observation of
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
ACh relaxation (%)
162
120
P=NS
80
40
0
OO XO+XX
Effect of GCH1 Haplotype on Vascular Superoxide,
Oxidative Stress, and Endothelial Function
The presence of the X haplotype was associated with higher total superoxide
(O2⫺) production (A) and greater L-NAME-inhibitable delta[O2⫺] (B) in human
saphenous veins. The X haplotype was also associated with significantly lower
plasma BH4:tBio ratio (C) and higher plasma levels of oxidized low-density
lipoprotein (ox-LDL) (D). In addition, the X haplotype was also associated with
lower maximum vasorelaxations in response to acetylcholine (ACh) (E) in
saphenous veins, although it had no impact on the vasomotor responses to
sodium nitroprusside (SNP) (F).
lower BH4:total biopterin ratio, and increased circulating
levels of oxidized LDL (Fig. 3).
To investigate how this GCH1 haplotype and the associated differences in biopterin levels modulate NOmediated endothelial function, we quantified the vasomotor
responses of vessel segments to ACh in an organ bath
system. Vasorelaxation responses to ACh were significantly
reduced in X haplotype carriers than in OO homozygotes,
whereas endothelium-independent vasomotor responses to
the direct NO donor, SNP, were identical between genotypes (Fig. 3).
Multivariate analysis. Because BH4 levels and endothelial
function may potentially be altered by many factors, we used
multivariate analysis to further investigate the relationships
between the GCH1 haplotype and biopterin levels, taking
into account other clinical and demographic factors. Presence of the X haplotype was an independent predictor of
reduced BH4 levels in both plasma and vascular tissue
(plasma: ␤ [SE] ⫺11.764 [4.722], p ⫽ 0.014; SV: ␤ [SE]
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
⫺3.06 [1.47], p ⫽ 0.039). Furthermore, presence of the X
haplotype was independently associated with increased vascular superoxide production (SV: ␤ [SE] 1.196 [0.573], p ⫽
0.04) and with reduced vasorelaxations to ACh (SV: ␤ [SE]
⫺5.304 [2.355], p ⫽ 0.026). Of the other additional
demographic and clinical factors, diabetes mellitus was
independently associated with increased vascular superoxide
production (␤ [SE] 1.520 [0.619], p ⫽ 0.016) and with
reduced ACh vasorelaxations (␤ [SE] ⫺5.442 [2.313], p ⫽
0.020), and hypercholesterolemia with reduced ACh vasorelaxations (␤ [SE] ⫺7.703 [2.236], p ⫽ 0.001). Female
gender was independently associated with reduced ACh
vasorelaxations (␤ [SE] ⫺7.646 [2.741], p ⫽ 0.006) and
with reduced BH4 levels (␤ [SE] ⫺5.24 [2.25], p ⫽ 0.022).
Discussion
In this study we have identified a striking effect of a novel
haplotype of the GCH1 gene on both plasma and vascular
BH4 levels in patients with CAD, mediated through altered
GCH1 expression. In turn, we have shown that these
genetically determined differences in BH4 availability are
independent and strong predictors of key features of
endothelial function, including aspects of eNOS coupling
such as vascular superoxide production and NO-mediated
vasorelaxation.
Tetrahydrobiopterin is an essential cofactor for eNOS
function in the vascular endothelium, with increasingly
recognized roles in the pathogenesis of vascular disease
states through effects on eNOS coupling (1) in both the
vascular wall and in circulating endothelial progenitor cells
(31). However, little is known about the pathophysiologic
control of endothelial BH4 levels in humans. In disease
states such as atherosclerosis (32), diabetes mellitus (17,31),
hypertension (12), or insulin resistance (33,34), vascular
BH4 deficiency seems to be mediated, at least in part, by the
increased intracellular oxidation of BH4 to BH2 and B by
reactive oxygen species (such as ONOO⫺) (7,32). However,
other studies suggest that changes in BH4 biosynthesis,
through regulation of the rate-limiting enzyme GTPCH
(35), may also be an important mechanism regulating
vascular BH4 bioavailability (34,36). The GCH1 gene,
encoding GTPCH, is expressed in several cell types, such as
macrophages (37), endothelial cells (38), and others. However, it has been unclear to what extent altered GCH1
expression in the human vascular wall is related to differences in vascular BH4 levels, or to aspects of endothelial
function in subjects with vascular disease. We have now
used the genetic strategy of Mendelian randomization in
humans to investigate the functional effects of intrinsic
differences in vascular GCH1 expression.
The importance of GCH1 expression and GTPCH enzymatic activity in regulating BH4 availability is well established. Loss-of-function mutations of GCH1 cause severe
BH4 deficiency, resulting in diseases such as doparesponsive dystonia (OMIM 128230) and variant phenyl-
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
163
ketonuria (OMIM 233910), because of dysfunction of the
BH4-dependent enzymes tyrosine hydroxylase and phenylalanine hydroxylase. Experimental mouse models with alterations in systemic or vascular-specific GCH1 expression (10,16) have shown that GTPCH is a key regulator
of vascular BH4 levels in vivo. For example, the hph-1
mouse has an ENU mutant allele that maps to the GCH1
locus, but without mutations in the either the GCH1 coding
sequence or minimal promoter (10). The hph-1 allele
results in reduced GCH1 expression and reduced BH4
levels, an intermediate phenotype in heterozygote animals and functional defects in both neurotransmitter and
NO synthesis (16).
We have recently shown that a specific GCH1 haplotype
(whose presence we define as X and absence as O) confers
significant reduction in the induction of GCH1 expression
and biopterin levels in leukocytes after a forskolin or
lipopolysaccharide challenge (18,19). Furthermore, this
GCH1 haplotype is associated with changes in pain perception and the severity of postoperative pain, likely because of
differences in GCH1 expression after nerve injury (18,19). In
addition, systemic inflammation seems to be a key regulator
of biopterin synthesis in patients with CAD because plasma
biopterins are correlated with plasma CRP (39). We have
now shown that the presence of the X haplotype is associated with lower levels of both plasma BH4 and total
biopterins (which is the sum of BH4, BH2, and B,
reflecting the overall biosynthetic activity of GTPCH) in
patients with multivessel CAD. These patients represent a
model of moderate inflammatory stimulation (mean CRP
2.2 mg/l, with range from 0.12 to 15.5 mg/l), consistent
with the effect of the haplotype on the regulation of GCH1
expression in white blood cells in response to lipopolysaccharide exposure. This effect was observed not only in the
uncommon XX genotype (2% of our population), but also in
the X haplotype carriers (XO genotype; 27.4% of the
population), suggesting that the X haplotype may be an
important and common factor in regulating circulating
biopterin levels in patients with CAD. Importantly, we
observed that the effect of the X haplotype on plasma BH4
was paralleled by an effect on vascular BH4, and on GCH1
mRNA levels in vascular tissue. It seems most likely that the
functional SNP or SNPs in the GCH1 X haplotype block
will have effects on GCH1 expression, for example through
effects on transcription, or on GCH1 mRNA stability and
translation. However, we have no evidence that any of the
SNPs used to define the GCH1 X haplotype have any direct
functional effects. Indeed, the SNPs chosen to screen the
GCH1 gene were not selected on the basis of possible
functional importance. Further recent evidence suggests
that another SNP, located in the 3= UTR of the GCH1
gene, is also associated with reduced biopterin-dependent
effects (40). Whether this SNP is part of the same or
another haploblock as the X haplotype identified in our
studies remains to be seen. Further studies will be required
to ascertain which component SNP or SNPs confer the
164
Antoniades et al.
GCH1 Haplotypes and Vascular Biopterins
functional effects on GCH1 expression and how such an
effect might be mediated.
Our observations suggest that GCH1 haplotype plays a
potentially important role in regulating vascular BH4 bioavailability in CAD. More generally, our results highlight
the central role of GTPCH, and GCH1 gene expression in
particular, as key regulators of biopterin availability in
humans, and in the functional responses of the vascular
endothelium in vascular disease states. In the vascular
endothelium, BH4 mediates coupling of oxygen reduction
to heme-catalyzed L-arginine oxidation to form NO and
L-citrulline (2). Therefore, BH4 deficiency is proposed to
lead to eNOS uncoupling, resulting in decreased NO
bioavailability and increased production of superoxide radicals from the uncoupled enzymatic form (41). A BH4
deficiency is associated with eNOS uncoupling in experimental models (17), resulting in increased superoxide production and decreased NO bioavailability in the vascular
wall (41). Indirect evidence from previous clinical studies
suggested that increased BH4 bioavailability is associated
with improved endothelial function and eNOS coupling
(5,6). However, the impact of endogenous BH4 levels on
vascular function in patients with vascular disease has
remained uncertain.
The identification of a GCH1 haplotype with a major
effect on endogenous BH4 levels in patients with CAD
provided us with a novel means to test the functional effect
on BH4 in the vascular wall. Indeed, we observed that the
presence of the X haplotype was associated with both higher
vascular superoxide and a greater L-NAME inhibitable
fraction of superoxide, representing superoxide derived from
uncoupled eNOS. This finding supports the hypothesis that
vascular BH4 regulates eNOS coupling in vessels from
patients with CAD. The impact of the X haplotype on
vascular redox state was also reflected in the systemic levels
of ox-LDL and the BH4/total biopterin ratio, both systemic
biomarkers of the overall oxidative stress status. To further
evaluate the impact of the X haplotype on eNOS function,
we examined its effect on the vasomotor responses of the
same vessels to acetylcholine, observing that the X haplotype
was associated with reduced vasorelaxations to acetylcholine,
implying that it has a direct impact on NO bioavailability and
endothelial function in these vessels. Importantly, we observed
that the effects of this haplotype on vascular BH4, superoxide
production, eNOS coupling, and endothelial function were
independent of the presence of other risk factors for
atherosclerosis, suggesting that this haplotype may prove to
be an independent risk factor for atherosclerosis.
Study limitations. Some limitations of the present study
need to be acknowledged. We were unable to evaluate the
effect of GCH1 haplotypes in vessels from healthy individuals because these measurements require surgical material
(segments of SV and IMA grafts used in CABG) that is not
available from healthy individuals. We evaluated endothelial
function in isolated SV segments, rather than arterial
segments, because of the limited quantity of arterial tissue
JACC Vol. 52, No. 2, 2008
July 8, 2008:158–65
available from IMA grafts. However, we have used SV in
the past as a reliable model system for the study of human
vascular endothelium (5,6,20). Finally, our use of lucigeninenhanced chemiluminescence provides only a measure of
overall vascular superoxide generation rather than identifying the specific cell types involved.
Conclusions
In conclusion, we have shown that a novel haplotype of the
GCH1 gene is a major determinant of BH4 bioavailability in
both plasma and vascular endothelium in patients with
CAD. Our findings show that GCH1 expression is a key
regulator of vascular biopterin levels, and in turn an important determinant of vascular oxidative stress and endothelial
function. Genetic differences in BH4 availability could have
potentially important effects on vascular disease pathogenesis, and provide a means to test the effects of BH4 on
clinical outcomes and cardiovascular risk, a question that
will need to be addressed in large case-control and prospective follow-up studies.
Reprint requests and correspondence: Dr. Keith M. Channon,
Department of Cardiovascular Medicine, University of Oxford,
John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
E-mail: [email protected].
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Key Words: tetrahydrobiopterin y endothelial nitric oxide synthase y
GTP-cyclohydrolase I y superoxide y haplotype y atherosclerosis.