Long-Term Administration of Endothelin Receptor Antagonist Improves Coronary Endothelial Function in Patients With Early Atherosclerosis
Background— Endothelin (ET-1) is one of the most potent vasoconstrictors and plays a seminal role in the pathogenesis of atherosclerosis. The present study was designed to test the hypothesis that long-term treatment with an endothelin-A (ETA) receptor antagonist improves coronary endothelial function in patients with early coronary atherosclerosis.
Methods and Results— Forty-seven patients with multiple cardiovascular risk factors, nonobstructive coronary artery disease, and coronary endothelial dysfunction were randomized in a double-blind manner to either the ETA receptor antagonist atrasentan (10 mg) or placebo for 6 months. Coronary endothelium-dependent vasodilation was examined by infusing acetylcholine (10−6 to 10−4 mol/L) in the left anterior descending coronary artery. NG-monomethyl-l-arginine was administered to a subgroup of patients. Endothelium-independent coronary flow reserve was examined by use of intracoronary adenosine and nitroglycerin. Baseline characteristics and incidence of adverse effects were similar between the 2 groups. There was a significant improvement in percent change of coronary blood flow in response to acetylcholine at 6 months from baseline in the atrasentan group compared with the placebo group (39.67%, 95% confidence interval 23.23% to 68.21%, versus −2.22%, 95% confidence interval −27.37% to 15.28%; P<0.001). No significant difference in the percent change of coronary artery diameter or change in coronary flow reserve was demonstrated. Coronary blood flow, coronary artery diameter, and the effect of NG-monomethyl-l-arginine were similar between the groups at baseline and at 6 months.
Conclusions— This study demonstrates that 6-month treatment with atrasentan improves coronary microvascular endothelial function and supports the role of the endogenous endothelin system in the regulation of endothelial function in early atherosclerosis in humans.
Clinical Trial Registration Information— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00271492.
Received November 19, 2009; accepted June 16, 2010.
Endothelin-1 (ET-1) is a 21–amino acid peptide with both mitogenic and vasoconstricting properties.1 Circulating ET-1 is increased in patients with atherosclerosis2 and in the coronary circulation of patients with early atherosclerosis and coronary endothelial dysfunction.3 ET-1 contributes to the complex regulation of vascular tone through 2 major receptors, termed endothelin-A (ETA) and endothelin-B (ETB) receptors.4 ETA receptors are located on vascular smooth muscle and mediate vasoconstriction.5 ETB receptors are located on both endothelial cells, where they mediate dilation by releasing nitric oxide (NO), and on smooth muscle cells, where they contribute to constriction.6
Clinical Perspective on p 966
Short-term blockade of ETA receptors results in coronary vasodilatation7,8 and improvement in endothelial function.7,9 We have previously reported that 12-week administration of an ETA receptor antagonist in a porcine hypercholesterolemia model improved coronary endothelial function.10 Atrasentan (ABT-627, A-147627; trade name Xinlay; Abbott Laboratories, Abbott Park, Ill), an orally available, potent, and highly selective antagonist of the ETA receptor,11 has been tested extensively in cancer therapy, and its effects have been reported in several phase III trials of refractory malignancies.12,13 We have recently demonstrated the systemic effects and the safety of long-term administration of atrasentan in humans.14 The effect of long-term therapy with an ETA receptor antagonist on the human coronary circulation is unknown. Thus, the present study was designed to extend our previous observations and to test the hypothesis that long-term administration of an ETA receptor antagonist improves coronary endothelial function in patients with early coronary atherosclerosis. Moreover, we evaluated the impact of therapy on coronary NO bioavailability.
This study is a single-center, double-blind, randomized controlled trial sponsored by the National Institutes of Health. The study protocol was approved by the Mayo Foundation Institutional Review Board.
Subjects were enrolled between July 2001 and December 2006 from those referred to the cardiac catheterization laboratory for evaluation of coronary artery disease who were found to have nonobstructive disease and who had a comprehensive coronary physiology study that included the assessment of endothelial function and non–endothelium-independent coronary flow reserve (CFR). Patients were included in the present study if they had coronary microvascular endothelial dysfunction. According to our previous studies, we defined microvascular endothelial dysfunction as a ≤50% increase in coronary blood flow (CBF) in response to the maximal dose of acetylcholine (ACh) compared with baseline CBF.15 Exclusion criteria for the study have been reported previously.15
At baseline, diagnostic coronary angiography and determination of endothelium-dependent and -independent flow reserve were performed as described previously.15 A Doppler guidewire (0.014-in diameter, FloWire, Volcano Corp, San Diego, Calif) within a 2.2F coronary infusion catheter (Ultrafuse, SciMed Life System, Maple Grove, Minn) was advanced and positioned in the middle portion of the left anterior descending coronary artery. Intracoronary bolus injections of incremental doses (18 to 36 μg) of adenosine (Fujisawa Pharmaceutical Co, Osaka, Japan), an endothelium-independent vasodilator (primarily of the microcirculation),16 were administered into the guiding catheter until maximal hyperemia was achieved.
Assessment of endothelium-dependent CFR was performed by selective infusion of ACh into the left anterior descending coronary artery. ACh (Iolab Pharmaceuticals, Claremont, Calif) 10−6, 10−5, and 10−4 mol/L was infused at 1 mL/min for 3 minutes.3,15 Hemodynamic data (heart rate and mean arterial pressure), Doppler measurements, and coronary angiography were obtained after each infusion. Endothelium-independent epicardial vasodilation was assessed with an intracoronary bolus injection of nitroglycerin (200 μg, Abbott Laboratories).17
Assessment of Tonic Basal NO Release
In a subset of patients whose consent was obtained before the baseline comprehensive coronary physiology study (some patients provided consent after their initial cardiac catheterization and did not have this part of the study performed), intracoronary NG-monomethyl-l-arginine (L-NMMA), a specific inhibitor of NO synthesis, was infused at a rate of 32 μmol/min for 5 minutes and then at 64 μmol/min for another 5 minutes. Basal NO activity was evaluated by measuring the effect of L-NMMA on the percent change in coronary artery diameter and the percent change in CBF.
Quantitative Coronary Angiography
Coronary artery diameter was analyzed from digital images by use of a modification of a previously described technique from this institution.15 The left anterior descending coronary artery was divided into proximal, middle, and distal segments. For each segment, the measurements were performed in the region where the greatest change had occurred during the ACh infusion. An angiographically smooth segment of the proximal, middle, and distal left anterior descending coronary arteries, free of any overlapping branch vessels, was identified in each patient and served as the reference diameter for calculation of diameter stenosis. End-diastolic cine frames that best showed the segment were selected, and calibration of the video and cine images was accomplished with the diameter of the guide catheter identified. Quantitative measurements of the coronary arteries were obtained with a computer-based image-analysis system. Segment diameters were determined at baseline and after both ACh and nitroglycerin administration. The proximal segment was not exposed to Ach and thus served as a control segment.
Assessment of CBF
Doppler flow-velocity spectra were analyzed online to determine the time-averaged peak velocity. Volumetric CBF was determined from the following relation: CBF=cross-sectional area×average peak velocity×0.5.18 Endothelium-dependent CFR was calculated as percent change in CBF in response to ACh, as described previously.19 The endothelium-independent CFR ratio was calculated by dividing the average peak velocity after adenosine injection by the baseline average peak velocity.15 Coronary vascular resistance (CVR) was estimated as mean arterial blood pressure divided by CBF.20
Patients were randomly assigned and treated in a double-blind fashion according to a computer-generated code with either the ETA receptor antagonist atrasentan at a dose of 10 mg PO once per day or placebo for 6 months, in addition to standard medical therapy. Treatment assignments were concealed from participants and study staff except for the pharmacist technician. Study and placebo tablets (provided by Abbott Laboratories) were distributed in bottles and were identical in appearance.
The 6-month follow-up coronary artery angiogram with coronary physiology study was performed by an independent investigator blinded to treatment allocation. The prespecified primary end point was the percent change in CBF measured by intracoronary graded administration of ACh and the percent change in coronary artery diameter measured by quantitative coronary angiography at 6 months from baseline.
On the basis of our previous studies,21 and assuming a power of 80% and an α-error of 0.05, we calculated the magnitude difference that could be detected for the percent change in coronary artery diameter in response to ACh 10−4 mol/L as 3.9% and for the percent change in CBF in response to ACh 10−4 mol/L as 37.3% for a sample size of 70 (35 in each group). With the above-magnitude difference and given the observed placebo mean from our previous studies of −25.9% for percent change in coronary artery diameter in response to ACh 10−4 mol/L, we would expect a significant result if the treatment mean were greater than −22.0%. Similarly, given an observed placebo mean of 6.0% for percent change in CBF in response to ACh 10−4 mol/L from our previous studies, we would expect a significant result if the treatment mean were greater than 43.3%.
Data are displayed as mean±SD or count and percentage as appropriate. Variables with heavily skewed distribution are reported as medians with first and third quartiles in parentheses. Analysis to compare different demographic and baseline clinical data between the randomized groups was performed with the Student t test for continuous data and the Pearson χ2 test for categorical data. Baseline data for CBF, CFR, and coronary artery diameter were compared with a rank sum test. Differences between groups in the primary end points were compared with Wilcoxon rank sum test. Multiple linear regression was used to estimate the treatment effect adjusted for other covariates. All statistical tests were 2-sided, and P<0.05 was considered to be statistically significant.
Characteristics of the Patients
Of the 72 patients randomized, 47 had a repeated coronary artery angiogram with coronary physiology study at 6 months and were included in the final analysis (Figure 1). The baseline characteristics of the 47 subjects were similar between the atrasentan and placebo groups (Table 1). Briefly, the groups were well matched at baseline with regard to age, gender, race, coronary risk factors, and medical treatment.
The baseline characteristics of the entire cohort have been reported previously.14 The patients who had a follow-up coronary physiology study did not differ significantly from those who did not. Patients who had a follow-up coronary physiology study, however, had a greater use of calcium channel blockers and diuretics at baseline than those who did not have a follow-up physiology study (Table 2).
Blood Pressure and Heart Rate
The baseline heart rate and systolic and diastolic blood pressures were similar between the 2 groups. Long-term administration of atrasentan resulted in a reduction in diastolic blood pressure from 74±3 to 59±17 mm Hg (P=0.02) and in mean arterial pressure from 94±12 to 86±12 mm Hg (P=0.004), but the reduction in systolic blood pressure from 120±36 to 113±27 mm Hg was not significant (P=0.96). Systolic, diastolic, and mean arterial blood pressures did not change in the placebo group. There was no effect on heart rate (Table 3).
Effect of Atrasentan on Resting Coronary Vascular Tone
There was no difference in resting coronary artery diameter or CBF between the groups at baseline or at 6 months (Table 3).
Effect of Atrasentan on CVR
Long-term administration of atrasentan decreased CVR, although this did not reach statistical significance (P=0.08). CVR at 6 months was lower in the atrasentan group than in the placebo group (P=0.04). The difference in CVR from baseline to 6 months between the atrasentan and placebo group, however, was not significant (P=0.5; Table 3).
Endothelium-Dependent and -Independent Coronary Vascular Function
Effect of Atrasentan on the Epicardial Circulation
Analysis of percent change in coronary artery diameter in response to ACh in the middle and distal segments of the left anterior descending coronary artery produced similar results, and we report only the results for the middle segment. Epicardial response to ACh did not improve after long-term administration of atrasentan (Figure 2). The percent change in coronary epicardial diameter in response to ACh at 6 months was similar between the atrasentan group and the placebo group (P=0.26; Table 3).
Effect of Atrasentan on Coronary Microvascular Endothelial Function
Long-term administration of atrasentan resulted in significant improvement in coronary microvascular endothelial function (Figure 2). Compared with placebo, 6-month therapy with atrasentan resulted in a significant improvement in percent change in CBF in response to ACh (P=0.003). There was also a significant difference in improvement in percent change in CBF between the atrasentan and placebo groups at 6 months compared with baseline (P<0.001; Table 3). In adjusted analysis using a linear regression model, atrasentan compared with placebo significantly predicted improvement in coronary microvascular endothelial function even after adjustment for mean arterial pressure, glucose, triglycerides, lipoprotein(a), and uric acid (adjusted difference in the mean percent change in CBF=29.86, P<0.001).
Effect of Atrasentan on Coronary Endothelium-Independent Function
Endothelium-independent CFR in response to adenosine was lower with atrasentan (P=0.01). CFR in the placebo group also decreased, although this did not reach statistical significance (P=0.06). Intracoronary administration of adenosine produced a lower CFR in the atrasentan group at 6 months than in the placebo group (P=0.04); however, there was no significant difference in the change in CFR from baseline to 6 months between the atrasentan and placebo groups (P=0.64; Table 3).
Effect of Atrasentan in Response to Intracoronary Nitroglycerin
There was no difference in the percent change in coronary artery diameter after administration of intracoronary nitroglycerin between the groups at baseline or at 6 months (Table 3).
Effect of L-NMMA
A total of 30 patients (15 in each group) had L-NMMA administered at baseline and at 6 months. The effect of L-NMMA on percent change in coronary artery diameter and percent change in CBF was similar between the atrasentan and placebo groups at baseline and after 6 months of treatment (Table 3), which indicates similar blockade of tonic basal release of NO from the coronary circulation in both groups. The baseline characteristics of the patients who received the L-NMMA study were similar to those of the patients who did not, which suggests that they may be representative of the entire cohort.
Effect of Atrasentan on Renal Function and Metabolic Characteristics
Triglyceride level (P=0.013), lipoprotein(a) (P=0.046), uric acid (P=0.006), fasting blood glucose (P=0.026), and glycosylated hemoglobin (P=0.041) improved at 6 months in the atrasentan-treated patients compared with placebo-treated patients. Comparison of the difference at 6 months from baseline between the atrasentan-treated patients and placebo-treated patients, however, was significant only for fasting glucose (P=0.02). No significant differences in changes at 6 months in the creatinine level (P=0.25) or estimated creatinine clearance (P=0.09) were demonstrated between the groups (Table 4).
The incidence of reported adverse effects was similar between the treatment groups (Table 5). The most common adverse effect with atrasentan was nasal stuffiness, which occurred in the first week after initiation and persisted during the study period. Headache occurred with a higher incidence in patients receiving atrasentan in the first month but was reported at the same rate in the 2 groups on further follow-up. Edema (upper extremities and facial) occurred more frequently with the initiation of atrasentan, but after 2 months of follow-up, there were no differences between the groups.
There were no changes in body weight in the patients treated with atrasentan. There were no changes in levels of sodium or albumin in the atrasentan group. No patient developed proteinuria or hematuria during the study period.
A mild drop in hemoglobin concentration was observed within the first month of treatment but remained stable within the subsequent 5 months. In the atrasentan group, the reductions in mean hemoglobin at the end of the treatment were 1.18±1.17 g/dL compared with 0.63±0.90 g/dL in the placebo group (P=0.04); no patient required blood transfusion during the study period. No significant changes were observed in white blood cell count or platelet count in the atrasentan-treated patients. There were no increases and no clinically significant changes in liver enzymes.
The present study demonstrates that long-term ETA receptor antagonism improves coronary microvascular endothelial function in patients with early atherosclerosis and nonobstructive coronary artery disease. ET-1– mediated ETA receptor activation, however, did not have any significant effect on resting coronary vascular tone or epicardial endothelial function. We have also previously shown improvement in the systemic hemodynamic and metabolic profile with long-term ETA receptor antagonism in humans.14 The present study serves as a natural extension of this study and supports a role for ET-1 in the regulation of coronary endothelial function in humans.
Effect of ETA Receptor Blockade on Coronary Vascular Tone
The present study did not show a significant contribution of ET-1–mediated ETA receptor activation to either the basal epicardial or microcirculatory coronary vasoconstrictor tone. Previous studies have demonstrated that acute blockade of ETA receptors resulted in dilation of coronary epicardial arteries.7,8 The effects on the coronary microcirculation, however, were much less pronounced,9 with some studies showing no change in CBF velocity after acute ETA receptor blockade.22 In 1 study, vasodilation by ETA receptor antagonism was greater in atherosclerotic coronary arteries than in normal coronary arteries,22 which reflects the relative increase in vascular tissue and circulating ET-1 concentrations in atherosclerotic plaques.2,23,24 Thus, the endogenous ET-1 pathway may play a more significant role in regulation of vascular tone in pathophysiological states associated with more advanced atherosclerotic plaques.2,15 Therefore, the lack of an effect of ETA receptor antagonism on basal coronary vascular tone in the present study may be related to the fact that the patient population did not have significant coronary atherosclerosis.
Effects of ETA Receptor Blockade on Microcirculatory Coronary Endothelial Function
Short-term ETA receptor antagonism causes improvement in microcirculatory coronary endothelial function.7,9 One study showed an improvement in coronary microcirculation function in response to ACh infusion, with the greatest impairment of endothelial function deriving the greatest improvement after acute ETA blockade.9 Recent studies have also shown that ETA receptor activation contributes to peripheral endothelial dysfunction.25 The present study is in agreement with these clinical observations and demonstrates for the first time the beneficial effects of long-term administration of an ETA receptor antagonist on microcirculatory coronary endothelial function.
In the present study, long-term ETA antagonism did not affect epicardial vessel endothelium-dependent or -independent function. Previous studies with acute ETA antagonism showed improvement in epicardial coronary endothelial function only in those segments that constricted in response to ACh administration.9 The present study cohort included both coronary vessels that constricted and those that dilated, with ACh likely attenuating the vasodilatory effect of atrasentan on the epicardial vessels.
Effect of ETA Receptor Blockade on Coronary Endothelium-Independent Function
Long-term ETA receptor antagonism decreased endothelium-independent CFR in response to adenosine. CFR is calculated as the ratio between hyperemic blood flow and resting CBF. The reduction in CFR in response to adenosine may thus reflect the relatively small increase in resting CBF (although not significant) or a reduction in CVR in response to long-term ETA receptor blockade.
Long-term ETA receptor antagonism may improve coronary microvascular endothelial function through several potential mechanisms, including direct effects on the vasoconstriction caused by ET-1 activity, a decrease in oxidative stress and inflammation, an improvement in metabolic characteristics, an attenuation of atherosclerosis, and augmentation of NO pathways.
Direct Effect on Vasoconstriction Activity of ET-1
The sustained and potent vasoconstrictive response of ET-1 is mediated primarily through activation of ETA receptors,24 which are the predominant receptors in vascular smooth muscle cells. In the present study and in previous studies of acute inhibition of ETA receptor in experimental porcine hypercholesterolemia,26 we did not show any attenuation in ACh-induced epicardial vasoconstriction. We previously reported the beneficial effects of atrasentan in lowering blood pressure observed in this study.14 Other clinical trials have also shown similar beneficial effects of ETA receptor blockade on systemic blood pressure.27 The improvement in blood pressure observed in the present study may have accounted for some of the improvement in the endothelial vasodilator function. However, in the adjusted analysis, the improvement in microvascular endothelial function occurred even after adjustment for the hemodynamic and metabolic effects of atrasentan. This suggests that attenuation of the vasoconstricting effect is not the only mechanism of improving endothelial function.
ET-1 and NO
Classically, endothelial dysfunction has been considered to be the result of a decrease in NO bioavailability.3 Deficiency of the endothelium-derived NO is well documented in coronary endothelial dysfunction28,29 and in human atherosclerotic arteries.24 ET-1 can decrease NO bioavailability by decreasing its production via inhibition of endothelial NO synthase activity or by increasing its degradation via formation of oxygen radicals.30,31 ET-1 also functionally offsets the vasodilator action of NO and thereby participates in regulation of vascular tone.
The similar response to L-NMMA in the atrasentan and placebo groups in the present study implies that the effect on basal NO production by ETA receptor blockade is also not the main mechanism in the improvement of microvascular endothelial function in patients with early atherosclerosis. The interaction between ET-1 and NO may involve the degradation of NO. We have previously demonstrated the association between oxidative stress and endothelial dysfunction in animal models.29,32 Diet-induced hypercholesterolemia in porcine experimental models resulted in blunted endothelial function in the renal circulation29 and in the coronary circulation,32 and these were restored by antioxidant interventions. We have also recently shown that that coronary endothelial dysfunction in humans is characterized by local enhancement of oxidative stress.33 Both ETA and ETB receptors can increase production of reactive oxygen species,34,35 which react with NO to produce peroxynitrite. ETA receptor blockade may lead to reduced production of reactive oxygen species and decreased NO degradation, leading to improvement in NO-dependent vasorelaxation.
Attenuation of Atherosclerosis
ET-1 is increased in patients with atherosclerotic risk factors such as hypertension, diabetes, and smoking. It is also elevated locally in atherosclerotic plaques,23,24 and the circulatory and vascular tissue levels correlate with the severity of atherosclerotic lesions.2,36 One of the mechanisms by which long-term ETA receptor antagonism improved coronary microcirculatory endothelial function may be by a reduction in risk factors that may contribute to endothelial dysfunction. We have reported that long-term treatment with atrasentan resulted in a reduction of blood pressure and an improvement in glucose and lipid metabolism, as well as an improvement in renal function, in a subset of patients not treated with angiotensin-converting enzyme inhibitors.14 Thus, the beneficial effect of blockade of the endogenous ET-1 pathway in the present study may be mediated in part by the reduction in blood pressure and improvement in lipid and glucose metabolism. By modifying atherosclerotic risk factors and attenuating the atherosclerotic process, long-term ETA receptor antagonists may attenuate the progression of atherosclerosis by reversing endothelial dysfunction.
There are several limitations to the present study. First, a significant number of patients did not have a follow-up coronary physiology study. Among these patients, some did not have a repeat angiogram, and the rest had a repeat angiogram but developed coronary artery vasoconstriction that precluded the endothelial function study. The baseline characteristics of the patients who had a follow-up coronary physiology study, however, did not differ significantly from those who did not. The rate of adverse effects was also similar between the 2 groups. Even with these measures, there remains a risk of bias in the results.
Second, the number of subjects having repeat coronary physiology studies was lower than expected, and this may have reduced the power to detect significant differences in some of the end points. Third, we did not perform an intention-to-treat analysis with regard to the primary outcome, because we did not have follow-up coronary physiology study data on a significant number of patients.
Fourth, the small sample size means that overfitting is a concern in the case of multiple regression modeling and may cause instability in model estimates. Overfitting is especially a concern when one is creating a prediction model with the intention of applying it to external data sets. In our case, the model was not aimed at predicting future outcomes but rather at controlling other factors in estimating the size of the atrasentan effect. Still, overfitting could result in instability of the effect estimate. We did not find this to be the case, however, because the adjusted atrasentan estimate was nearly the same as the unadjusted estimate, and the standard error was not so large as to render the hypothesis test nonsignificant. Thus, we are confident that our conclusions with regard to atrasentan are valid.
Fifth, the present study was not designed to explain the precise cellular mechanism of the effects of long-term administration of atrasentan on coronary physiology. Because of the lack of standardization in measurements of invasive endothelial function, we were limited in our ability to compare the observed effects with those from studies that measured more acute effects of atrasentan. The measured effects on coronary physiology may indeed reflect the acute effect from the last given dose(s). More studies are needed to elucidate the mechanisms of long-term ETA receptor antagonism in improving coronary microvascular endothelial function.
Finally, we did not have data on the degree of atherosclerosis in the patients through intravascular ultrasound. We thus could not correlate the improvement in endothelial function observed with the degree of atherosclerosis.
This study demonstrates that 6-month treatment with atrasentan improves coronary microvascular endothelial function. It emphasizes the importance of ET-1 in the cardiovascular system and the potential of long-term ETA receptor antagonism to improve endothelial dysfunction.
This study demonstrates for the first time that endogenous ET-1 plays a role in long-term coronary microvascular endothelial function and in the pathogenesis and progression of coronary endothelial dysfunction, a known prognostic factor for cardiovascular disease. The present study suggests a potential role for long-term ETA receptor antagonists as a therapeutic option for patients with coronary endothelial dysfunction and nonobstructive coronary artery disease.
Sources of Funding
The study was supported by grants from the National Institutes of Health (NIH K24 HL-69840, NIH R01 HL-63911, HL-77131, HL 92954, HL 085307, DK 73608, DK 77013) and the Mayo Foundation.
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Endothelial dysfunction is considered critical in the initiation, progression, and complications of coronary artery disease and is independently associated with cardiovascular events. It is a reversible process that represents the functional expression of an individual’s overall cardiovascular risk factor burden, and many therapies that restore endothelial function also decrease cardiovascular events. Coronary endothelial function is regulated by the balance of endothelium-derived vasodilator and vasoconstrictor factors such as endothelin-1. This study provides evidence that long-term ETA receptor antagonist administration improves coronary microvascular endothelial function in humans and supports a role for endogenous endothelin in the mechanism and potentially the treatment of coronary endothelial function in humans.