Tetrahydrobiopterin Improves Endothelial Function in... : Journal of Cardiovascular Pharmacology (original) (raw)
The endothelium regulates vascular tone by releasing vasoactive substances. One of these substances and probably the most important is nitric oxide (NO) (1). Biosynthesis of NO requires the activation of nitric oxide synthase (NOS) in the presence of tetrahydrobiopterin (BH4) as an essential cofactor (2,3). Biochemically BH4 directs flow of electrons toward enzyme-bound L-arginine. In addition BH4 stabilizes NOS in its active dimeric form and enhances substrate affinity of the enzyme for L-arginine (4).
Cardiovascular risk factors are associated with a reduced bioavailability of NO (5-8). The underlying defect may involve decreased formation (9) and increased degradation of NO by oxygen-derived free radicals (10). Previous reports from our group have shown that dysfunction of endothelial NOS may be a source of oxygen radical production (3,11). Activation of endothelial NOS in the absence of BH4 leads to an uncoupling of the L-arginine-NO pathway under in vitro conditions and causes in creased formation of oxygen-derived radicals (12). Therefore decreased availability of BH4 decreases NO production (9), whereas BH4 supplementation directly increases NO production and reduces superoxide generation (4).
Thus we hypothesized that, in patients with coronary artery disease, intracellular BH4 levels may be decreased compared with those in normal controls. This may shift the balance between NO and oxygen-free radicals toward endothelial dysfunction, which may induce and contribute to abnormal coronary vasomotion (see Fig. 1). If correct, this mechanism may lead to endothelial dysfunction and represent the basis for a new therapeutic strategy in patients with early atherosclerosis when NOS is not yet downregulated.
Schematic representation of NO synthase and influence of normal (top) and suboptimal (bottom) levels of tetrahydrobiopterin.
This study was designed to determine whether BH4 improves endothelium-dependent coronary vasomotion in patients with coronary artery disease.
PATIENTS AND METHODS
Patients
Nineteen patients with coronary artery disease who underwent coronary angiography for diagnostic purposes were included in this analysis (Table 1). Inclusion criteria were (a) angiographically normal branch of the left coronary artery (target segment), (b) percutaneous transluminal coronary angiography (PTCA) of another branch of the left coronary artery, and (c) written informed consent. The protocol was approved by the local ethical committee of the University Bern.
Patient characteristics (n = 19)
Study protocol
All vasoactive medication was stopped ≥24 h before catheterization. After diagnostic angiography, the culprit lesion was dilated without the use of vasoactive drugs (Fig. 2). The guidewire was left in place, and a 0.014 Cardiometrics FloWire was inserted into the target artery (angiographically normal branch). A baseline angiogram was acquired in optimal projections for this segment, and x-ray settings were fixed. Coronary diameters and coronary flow velocity reserve (18 μg adenosine, i.c., CFVR) were determined and repeated at each step of the study protocol. Next acetylcholine (ACh; 10−4_M_) was infused over a 2-min period via the guiding catheter. The BH4 (10−2_M_) was infused over a 2-min period, followed by a co-infusion of ACh, 10−4_M,_ and BH4, 10−2_M,_ for 2 min. Finally 300 μg i.c. nitroglycerin was administered.
Schematic representation of the study protocol. Angio, angiography; PTCA, percutaneous transluminal coronary angioplasty; ACh, acetylcholine (10−4 M, infused at 0.8 ml/min for 2 min); BH4, tetrahydrobiopterin (10−2 M, infused at 1 ml/min for 2 min); NTG, nitroglycerin (300 μg injected i.c.); CFVR, coronary flow velocity reserve.
Methods
(6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride (BH4; Alexis Corp., Läufelfingen, Switzerland) was prepared as a sterile solution using oxygen-free water (10−2_M;_ Hospital pharmacy, Inselspital Bern) to achieve intracoronary concentration of 10−4_M_ (assuming a blood flow of 160 ml/min for the left coronary artery). This dose was chosen according to previous studies, which showed maximal endothelial NO production at BH4 concentrations of 10−5-10−4_M_(13,14). ACh was diluted with NaCl, 0.9%, from sterile stock solution (Acetylcholine Dispersa; Ciba Vision Corp.) to a concentration of 10−4 (i.c., 10−6) M)(15,16).
Coronary angiography was carried out on a digital x-ray system (Philips DCI-SX and Philips Integris) at 12.5 frames/s. Simultaneous biplane projections were acquired in all patients, and rotation and angulation were adapted to minimize foreshortening of the target segment.
Quantitative evaluation (see Fig. 3) was carried out in biplane (n = 12) or monoplane projection (n = 7) when overlapping vessels were present. Measurements were performed using the ACA package on Philips DCI/Integris systems with an accuracy of <0.01 mm and a precision of <0.10 mm (17). Intraobserver variability was 0.11 mm, and interobserver variability, 0.10 mm (18). The tip of the guiding catheter (6 or 7F) was used for calibration purposes. The diameter of defined vessel segments was determined at baseline and at the various steps of the protocol. Care was taken to select vessels between two branching points and not to include branches. The same segment, identified by the anatomic landmarks, was assessed at all steps of the protocol. Target parameter was the reference diameter, which is derived from the diameter function of the whole vessel segment identified at start. Given a not considerably tapering vessel segment as chosen for this investigation (this can be assured by no or minimal difference between the so-called obstruction and reference diameter), this parameter represents the best approximation to the average diameter of the respective segment. Mean cross-sectional lumen area (CSA) was calculated from the two orthogonal views using an elliptical model. With monoplane data, a circular lumen was assumed. To ensure further the stability of the measurements, for each vessel segment, three measurements were carried out and averaged. Percentage changes were calculated in all patients using the baseline angiogram as reference.
Original recording of an angiogram after acetylcholine (left) and co-infusion of acetylcholine and tetrahydrobiopterin (right). Quantitative evaluation was performed with the ACA package (Philips system).
Coronary flow velocity was determined by a 0.014″ Doppler-tipped angioplasty guidewire (FloWire and FloMap; Cardiometrics, Inc.). Blood flow velocity was calculated by fast Fourier transformation from the Doppler frequency shift of a reflected 12.5-MHz signal.
By multiplying average peak velocity with coronary cross-sectional area shortly distal to the tip of the Doppler wire at the point of Doppler velocity measurement, a proportional value for absolute volume flow was calculated.
Coronary flow velocity reserve was calculated by dividing maximal flow velocity after injection of adenosine (18 μg) by resting flow velocity.
Statistics
Patient data are given as mean ± 1 standard deviation (SD), cross-sectional area and flow velocity measurements as well as calculated volume flows as mean ± 1 standard error of the mean (SEM). Statistical analysis was performed by an analysis of variance for repeated measurements (ANOVA) with Bonferroni correction for post hoc comparisons. Differences between measurements were considered significant at p < 0.05.
RESULTS
Effect of acetylcholine
Fifteen of the 19 patients showed coronary vasoconstriction (decrease of CSA by 18 ± 3% from 6.7 ± 1.0 to 5.6 ± 0.9 mm2; p < 0.0001) on ACh infusion. However, four patients had vasodilation (+39 ± 20%, NS) and thus no evidence of endothelial dysfunction. Data included in this analysis refer to those patients with vasoconstriction (endothelial dysfunction; Table 2, Figs. 4 and 5A and B), unless otherwise specified.
Calculated volume flows and relative flow changes at the Doppler measurement location
Changes in coronary cross-sectional area after infusion of acetylcholine (ACh 1), tetrahydrobiopterin (BH4), as well as after co-infusion of tetrahydrobiopterin and acetylcholine (BH4/ACh 2), and infusion of nitroglycerin (NTG) in 15 patients with endothelial dysfunction. Values expressed as mean ± SEM.
Average peak velocity (A) and coronary flow velocity reserve (B) at baseline and after infusion of acetylcholine (ACh 1), tetrahydrobiopterin (BH4), as well as after co-infusion of tetrahydrobiopterin and acetylcholine (BH4/ACh 2), and infusion of nitroglycerin (NTG) in 15 patients with endothelial dysfunction. Values expressed as mean ± SEM.
Baseline average peak velocity increased after ACh infusion from 19 ± 1 to 32 ± 4 cm/s in all patients (p < 0.001).
Calculated volume flow at the Doppler measurement location increased from 66 ± 10 to 88 ± 20 ml/min, NS. CFVR did not change.
Effect of tetrahydrobiopterin
Infusion of BH4 was associated with mild coronary vasodilation compared with baseline (CSA, 6.9 ± 1.0 mm2, +5 ± 3%, NS; Fig. 4). No significant change in average peak velocity and CFVR occurred. Volume flow amounted to 77 ± 12 ml/min.
Effect of co-infusion of acetylcholine and tetrahydrobiopterin
During co-infusion of BH4 and ACh, coronary CSA did not change in those 15 patients with vasoconstriction at first challenge (+2 ± 3% compared with baseline, p < 0.005 vs. ACh alone; Fig. 4). Coronary flow velocity increased after co-infusion of ACh and BH4 to 31 ± 4 cm/s (p < 0.01 vs. baseline, Fig. 5A). There was a trend toward an increased flow compared with ACh alone (110 ± 19 vs. 88 ± 20 ml/min), although ANOVA did not reach significance level because of the larger variations in this calculated parameter. CFVR decreased slightly (NS, Fig. 5B).
In the four patients with coronary vasodilation after ACh, the vasodilatory response persisted after coinfusion of ACh and BH4 (CSA, +33 ± 6%; p < 0.01).
Effect of nitroglycerin
Nitroglycerin induced marked coronary vasodilation (CSA. 8.5 ± 1.2 vs. 6.7 ± 1.0 mm2, +31 ± 5%; p < 0.001 vs. baseline; Fig. 4). Coronary flow velocity decreased, and CFVR increased (NS vs. baseline; Fig. 5A and B). However, CFVR was significantly higher after nitroglycerin (p < 0.01) when compared with ACh or ACh and BH4 co-infusion because of the opposite changes in resting flow velocity after the respective drugs. Volume flow amounted to 91 ± 23 ml/min.
DISCUSSION
This study demonstrates for the first time in patients with endothelial dysfunction that BH4 improves the response of the large epicardial coronary arteries to ACH infusion. This can be explained by an improvement in endothelial function and an increase in NO production, possibly together with antioxidant effects. Therefore substitution of this essential cofactor of NOS may be used to restore normal endothelial function in patients with coronary artery disease.
All patients in this study had significant stenosis of at least one major coronary artery. The target vessel was, however, an angiographically normal branch of the left coronary artery, which in 14 of the 19 patients elicited coronary vasoconstriction (endothelial dysfunction) after ACh provocation. BH4 supplementation during ACh infusion prevented this paradoxic vasoconstriction (Fig. 3). Hence, BH4 substitution in coronary arteries with endothelial dysfunction may improve the ability of the NOS system to produce NO in response to ACh administration. In addition, direct antioxidant properties of BH4 might have contributed to this reaction (4). The observation of a significant increase of epicardial coronary artery cross-sectional area when comparing measurements after ACh alone with those after co-infusion of ACh and BH4, in contrast to a nonsignificant augmentation of calculated volume flow, again argues in favor of a predominant action on the large epicardial vessels. This is in accordance with restoration of endothelial function by the NOS pathway. The basically unchanged CFVR further supports this conclusion. Tetrahydrobiopterin infusion by itself did not alter basal coronary artery size, which is in line with previous findings in the forearm circulation (13). BH4 by itself may exert both vasorelaxing (19) and vasoconstrictor effects (20) under in vitro conditions. A previous in vivo study in healthy volunteers reported vasodilator effects of intraarterial BH4 infusion in the forearm circulation (21). However, much higher doses of BH4 (32 mg/min) were used than in our study (3 mg/min).
ACh caused vasoconstriction of epicardial coronary arteries, in agreement with previous reports (16,22). Because in normal coronary arteries, ACh induces vasodilation through NO release (23), paradoxic vasoconstriction in atherosclerotic coronary arteries reflects reduced bioavailability of NO. This paradoxic response may be related to reduced synthesis of NO because of substrate deficiency (24), impaired signal-transduction pathways, or downregulation of NOS expression (25). Alternatively, reduced NO bioavailability may reflect increased breakdown by superoxide anions (26). NOS itself may be a source of superoxide anion production (11) Thus, BH4 may act by two potential mechanisms: First, shortage of BH4 shifts the balance between NO and oxygen free radicals toward the production of more radicals and, thus, may induce endothelial dysfunction. It has been shown that superoxide anion and hydrogen peroxide are produced during activation of isolated NOS at suboptimal concentrations of BH4(12). Furthermore, inhibition of BH4 synthesis reduced NO formation in endothelial cells (9). Thus optimal concentrations of BH4 are essential for an adequate NO production (3) Second, it has been shown that BH4 dose-dependently decreases superoxide generation by hypoxanthine/xanthine oxidase, suggesting a direct antioxidant effect of BH4, which may contribute to increased production of NO (4).
Limitations of the study
One possible limitation of the study is heterogeneity of risk factors for coronary artery disease in our study population. From the patients with vasoconstriction after ACh as indicator of endothelial dysfunction, only two of 15 had one risk factor, and 13 had two or more risk factors. All four patients with vasodilation after ACh had two or more risk factors. The pivotal criterion for inclusion in this study, cessation of all vasoactive medication 24 h before catheterization, necessitated an extensive screening, especially because our intervention policy is almost exclusively ad hoc PTCA, which necessitated "blinded" cessation of therapy. Therefore the numbers in this pilot study do not allow stratification for specific risk factors with regard to the impact on vasomotor response, nor was the study designed for this purpose. We have defined BH4 action in the human coronary circulation of patients with endothelial dysfunction as assessed by ACh-induced epicardial coronary vasoconstriction as the primary end point of the study. Our results might provide the rationale for assessing a considerably larger population with a less invasive technique, like positron emission tomography (PET) scans.
In summary, this study shows that substitution of BH4, an essential cofactor of NOS and a scavenger of oxygen-derived free radicals, is able to restore abnormal endothelium-dependent coronary vasomotion in response to ACh in patients with coronary artery disease. This may provide a new therapeutic approach for treatment of endothelial dysfunction.
Acknowledgment: This study was supported by a grant of the Swiss National Research Foundation (32-32541.91/2).
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Keywords:
Endothelial dysfunction; Coronary artery disease; Nitric oxide synthase (NOS); Coronary vasomotion; Tetrahydrobiopterin; Acetylcholine
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