Cross-sectional evaluation of brachial artery flow-mediated vasodilation and C-reactive protein in healthy individuals

Subodh Vermaa, Chao-Hung Wanga,f, Eva Lonnb, Francois Charbonneauc, Jean Buithieud, Lawrence M. Titlee, Marinda Fungc, Steve Edworthyc, Annette C. Robertsonc and Todd J. Andersonc,* for the FATE Investigators

a Division of Cardiac Surgery, Toronto General Hospital, Toronto, Canada
b Department of Medicine, McMaster University, Hamilton, Canada
c Cardiology Division, Department of Medicine, University of Calgary, 8th Floor, Foothills Hospital, 1403-29th Street NW, Calgary, Alta., Canada T2N 2T9
d Cardiology Division, McGill University, Montreal, Canada
e Division of Cardiology, Dalhousie University, Halifax, Canada
f Division of Cardiology, Chang Gung Memorial Hospital, Keelung, Taiwan

Received March 30, 2004; revised June 15, 2004; accepted June 24, 2004 * Corresponding author. Tel.: +1 403 944 1033; fax: +1 403 283 0744 (E-mail: todd.anderson{at}calgaryhealthregion.ca).


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
AIMS: The present study was designed to (a) examine the interrelationship between endothelial function and CRP in healthy individuals and (b) evaluate the relationship of each biomarker towards global Framingham risk scores.

METHODS AND RESULTS: Brachial artery flow-mediated vasodilatation (FMD), CRP, and traditional cardiovascular risk factors were measured in the Firefighters and Their Endothelium (FATE) study, which recruited 1154 male participants (mean age 47.4±9.8 years) with no known history of cardiovascular disease. No relationship was observed between FMD and CRP (p=0.96). FMD and the Framingham risk score tended to correlate but not significantly (p=0.07). A lower FMD was related to a higher systolic and diastolic blood pressure (p<0.001 and p=0.002, respectively) in the univariate analysis, and higher systolic blood pressure (p=0.001) in the multivariate analysis. Elevated CRP levels independently correlated most closely with overall Framingham risk score (r=0.36, p<0.001) and a weaker although statistically significant relationship was seen with individual traditional cardiovascular risk factors (p<0.005).

CONCLUSIONS: The current study provided evidence that brachial artery FMD had no relationship to CRP in a large cohort of healthy subjects. These observations suggest that the predictive value of CRP may be largely independent of abnormalities in endothelial function. The additive prognostic value of endothelial vasodilator testing remains to be established.


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Significant progress has been made in the prevention of coronary artery disease through the modification of traditional risk factors for atherosclerotic vascular disease (ASVD). Effective primary prevention requires a detailed assessment of risk in order to select patients for appropriate investigation and treatment. However, a significant number of subjects at high coronary heart disease risk do not have traditional risk factors.1 As such, researchers continue to explore new biomarkers of atherosclerotic risk. In recent years, C-reactive protein (CRP), a marker of inflammation, has received substantial attention as a promising biological predictor of ASVD.2,3 Likewise, endothelial function has also been suggested as an independent marker of ASVD.4,5 Although experimental studies6,7 and small clinical studies8–10 suggest that CRP is related to endothelial function, this relationship has not been investigated in a large cohort of healthy subjects.

Elevated levels of CRP are associated with atherosclerotic risk in a variety of populations and disease modalities,2,3 and offer predictive value exceeding that of LDL-cholesterol and Framingham risk assessment.11,12 Endothelial dysfunction has been observed in patients with ASVD risk factors even in the absence of evidence for atherosclerotic lesions13 and has been suggested to be a predictor of vascular events.14–16 The present study was designed to (a) examine the interrelationship between endothelial function and CRP in healthy individuals and (b) evaluate the relationship of each biomarker towards global Framingham risk.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Firefighters and Their Endothelium (FATE) study is a prospective, longitudinal study of active and retired firefighters across Canada designed to assess the value of brachial artery flow-mediated vasodilatation (FMD) to predict cardiovascular events. The details of objectives and design have been described elsewhere.17 Participants were not eligible if they had documented coronary artery disease, cerebral vascular disease, or peripheral vascular disease. The current report was based on the data from the initial 1154 subjects.

Brachial artery ultrasound for assessment of flow-mediated vasodilatation
Brachial artery FMD was first described by Celermajer5 and has been extensively used by all FATE investigators according to previously described methodology.4,18 A 7.5-MHz linear phased array ultrasound transducer attached to a SONOS 5500 ultrasound machine (Hewlett–Packard) was used to image the brachial artery longitudinally just above the antecubital fossa. The vascular tourniquet was placed on the upper arm in order to create reactive hyperaemia for this study. A common methodology was used at all sites, which was confirmed by a site visit from the head technologist from the co-ordinating site. Vasoactive medications were held for ⩾18 h before the study in all patients.

After baseline measurements of the brachial artery diameter were recorded, a blood pressure cuff was inflated on the proximal portion of the arm to 260 mmHg for 5 min, creating distal limb ischaemia. After release of the cuff, reactive hyperaemia occurs, that is, flow in the brachial artery increases to accommodate the dilated resistance vessels in the forearm. The brachial artery was imaged for the first 2 min of reactive hyperemia. The flow-mediated dilator response was used as a measure of endothelium-dependent vasodilation. The brachial artery diameter was allowed to return to baseline level (~5 min after cuff release). Then, 0.4 mg of nitroglycerin was given sublingually, and the brachial artery was imaged for the ensuing 3 min to measure peak diameter. The response to nitroglycerin is a measure of endothelium-independent vasodilation.

Analysis
FMD analysis was performed at the core laboratory in Calgary by a single technician with extensive experience in ultrasound analysis. Two sequential diastolic frames (taken at the R wave on the ECG) for baseline, reactive hyperemia, repeat baseline and nitroglycerin stages were digitized. Straight segments of the artery (10 mm in length) were chosen. Computer-assisted edge detection brachial artery analysis software (DEA, Montreal, Canada) was used to calculate brachial artery diameters. The two frames were then averaged for each phase. Endothelium-dependent FMD was defined as the maximal percent change in brachial artery diameter (between 60 and 90 seconds) after reactive hyperaemia compared to baseline. The intra-observer and inter-observer variability for repeated measurements were 0±0.07 mm and 0.05±0.16 mm, respectively, in our laboratory.4 To document reproducibility of the measurement, 50 subjects had repeat FMD testing on a second occasion, 6–12 months following the baseline evaluation. While the group mean was the same on both occasions (8.2±3.2% vs. 8.3±2.8%), the mean of the absolute difference between determinations for each subject was a very favorable 1.8±1.6%.

Risk factor definitions
The following definitions were used for categorical assignment of risk factors: hypercholesterolemia – total cholesterol >5.2 mmol/L; hypertension – documented blood pressure on two occasions >140/90 mmHg; smoking – current cigarette smoking; family history – first degree relative with atherosclerotic vascular disease <60 years of age; diabetes mellitus – fasting blood glucose >7.0 mmol/L. Framingham risk scores were calculated as previously suggested by Grundy et al.19

High sensitive-CRP measurements
Fasting blood samples of plasma were obtained at baseline and stored at –70 °C. CRP concentrations were measured by a particle-enhanced immunoturbidimetric method with the use of an Hitachi 912 analyser (Roche Diagnostics) and reagents of Tina-quant C-reactive protein [latex] ultra sensitive assay (Roche Diagnostics). This measurement was standardized against the International Federation of Clinical Chemistry Certified Reference Material Standard (IFCC CRM 470). The lower detection limit reported for the assay was 0.21 mg/L and the co-efficient of variation at 0.21 mg/L was an acceptable 7.2%.

Statistical analysis
The data are expressed as the mean value±SD. Because CRP levels have a rightward skewed distribution, natural logarithmically transformed CRP values were used to relate FMD percentage and other variables. The data distribution of each variable was shown according to the quartiles of both FMD percentage and CRP levels. In addition, data analysis was also performed based on CRP levels of <1, 1–3, <3 and ⩽10, and >10 mg/L as suggested by the American Heart Association.20 Bivariate correlation (Spearman correlation) was utilised to evaluate the continuous relation between cardiovascular risk factors and CRP or FMD in the whole cohort and in subgroup analyses. Multivariate analysis was performed with backward stepwise linear regression model with controlling for potential confounders and known cardiovascular risk factors. In order to determine if there were site-specific differences in the relationship between FMD and other variables, the analysis was carried out separately for individual sites. No differences were noted between sites (data not shown), and as such the reported data represents the entire cohort. All analyses were performed with SAS software Version 8. Statistical significance was defined as a two-sided p value <0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population
The study population consisted of 1154 active and retired male fire-fighters, with no previous history of atherosclerotic vascular disease, enrolled from five medical centres in Canada as part of the FATE study.17 Table 1 shows the demographics of the study participants. There were 41.2%, 43.9% and 14.8% of subjects with CRP<1, 1–3 and >3 mg/L, respectively.


View this table:
[in this window]
[in a new window]
 
Table 1. Demographics
 
Variables and cardiovascular risk factors related to elevated CRP levels
Table 2 shows the data of each variable according to the quartiles of CRP concentrations. In univariate analysis, higher CRP concentrations were significantly correlated to older age (r=0.26), higher body mass index (r=0.144), higher systolic and diastolic blood pressure (r=0.21 and 0.16, respectively), higher total and LDL cholesterol (r=0.15 and 0.14, respectively), triglyceride ( r=0.13), fasting glucose concentrations (r=0.17), Framingham scores (r=0.36), smoking (r=0.06), and lower HDL cholesterol concentrations (r=–0.15). However, there was no substantive correlation between CRP levels and brachial artery FMD (p=0.957). All individual Framingham risk factors and body mass indexes were further analysed by multivariate model, which showed that age, total cholesterol, systolic blood pressure, high density lipoprotein, smoking and body mass index were all independent predictors of elevated CRP levels (Table 3). Body mass index added to the individual variables in Framingham Risk Scores (r value from 0.36 to 0.44).


View this table:
[in this window]
[in a new window]
 
Table 2. Distribution of brachial flow-mediated dilatation, and cardiovascular risk factors according to quartiles of baseline plasma concentrations of C-reactive protein
 

View this table:
[in this window]
[in a new window]
 
Table 3. Multivariate analysis for body mass index and individual variables in Framingham risk scores independently correlated with high sensitive C-reactive protein
 
Relationship between flow-mediated dilatation and cardiovascular risk factors
Table 4 shows the data of each variable according to the quartiles of brachial artery FMD percentage. In univariate analysis, a lower FMD correlated to a higher systolic and diastolic blood pressure (r=–0.1 and –0.09, respectively), and had a borderline correlation with older age and higher Framingham scores (Fig. 1, right panel). FMD was not related to the CRP concentrations (Fig. 1, left panel), body mass index, lipid profiles, fasting glucose concentrations and smoking status. However, when subjects within the lowest quartile of FMD (<5.8%) were analysed, a lower FMD was weakly but significantly related to higher diastolic blood pressure (p=0.03, r=–0.13), total and LDL cholesterol levels (p=0.02, r=–0.13 and p=0.02, r=–0.13, respectively), Framingham risk scores (p=0.04, r=–0.12) and CRP concentrations (p=0.01, r=–0.15). In multivariate analysis for the whole study population, only systolic blood pressure was significantly related to FMD (p=0.001).


View this table:
[in this window]
[in a new window]
 
Table 4. Distribution of C-reactive protein, cardiovascular risk factors according to quartiles of brachial flow-mediated dilatation
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1 Correlations of brachial artery flow-mediated vasodilatation (FMD) with C-reactive protein levels (a) and Framingham risk scores (b). % risk, 10-year risk for coronary heart disease.

 
Correlation of CRP and FMD in subgroups
Since CRP levels exceeding 10 mg/L may indicate the presence of occult infection or other systemic inflammatory processes,20 we also performed an analysis on the cohort after excluding subjects with CRP levels greater than 10 mg/L. There were 26 (2.3%) subjects with CRP levels higher or equal to 10 mg/L. Even after exclusion of these patients, no relationship between CRP and FMD could be elucidated.

The relationship between CRP and FMD was evaluated in subjects with specific cardiovascular risk factors. There was no significant correlation between CRP levels and forearm FMD in subgroups, except for the subgroup of active smokers. CRP levels in smokers were higher than non-smokers (1.96±1.85 mg/L vs. 1.57±1.39 mg/L, p=0.02). In smokers (n=128), univariate analysis showed a borderline relationship between a higher CRP and a lower FMD (p=0.12). After adjustment for age, blood pressure, lipid profiles, and glucose levels, a lower FMD significantly correlated to a higher CRP level (p=0.04).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our study indicates that brachial artery FMD had no relationship with CRP and was weakly associated with traditional coronary risk factors among this large cohort of healthy subjects. Although the prognostic value of endothelial function testing remains to be confirmed in large-scale trials, these data suggest that endothelial function testing may provide information distinct from CRP and traditional risk factors.

Dysfunction of endothelial cells is probably the earliest event in the process of lesion formation, and hence the concept that assessment of endothelial function may be a useful prognostic tool for coronary artery disease.21 While the ideal methodology for assessing endothelial function has not been established, there is significant evidence to suggest that brachial artery ultrasound provides an adequate assessment of endothelial health.22 FMD can be reproducibly measured by experienced technicians in a research environment as documented in the current study and this measurement has been shown to be nitric oxide-dependent. Moreover, measurement of the brachial artery reactivity may reflect the health of the coronary endothelial function.4 Interventions known to decrease cardiovascular mortality, such as statins and angiotensin converting enzyme inhibitors, also improve endothelium-dependent vasodilation.18 A number of studies in both the coronary15 and peripheral circulation16 have evaluated the prognostic value of endothelial function.23

The present study demonstrated a poor correlation between FMD and traditional coronary risk factors. Importantly, previous studies relating FMD and risk factors have yielded mixed results and have suffered from small sample sizes, different methodology, and heterogeneous populations. In the largest study to date, Celermajer and colleagues assessed FMD in 500 healthy subjects. In multivariate analysis there was no relationship between lipid parameters or blood pressure and FMD. The strongest determinants were age and cigarette smoking.24 In a population of young adults (n=326), Leeson et al.25 demonstrated a weak relationship between n–3 fatty acids and FMD in certain subgroups but there was absolutely no relationship with standard risk factors. In the present study there was a weak relationship between FMD and blood pressure, age and Framingham risk scores in a univariate model and only systolic blood pressure in multivariate analysis. Given the large sample size of the current analysis, one should conclude that FMD in a healthy population is not closely related to traditional risk factors. FMD could represent a potentially unique barometer of vascular health in which the effects of the traditional risk factors are modified by various factors that are not currently measured or appreciated.

An alternate hypothesis is that brachial artery FMD is not a good measure of endothelial function and as such does not correlate with traditional risk factors or CRP. The total weight of evidence would suggest that brachial artery FMD is a good measure of endothelial function despite the lack of correlation with risk factors or CRP. It may be warranted that repeated assessments of FMD can identify the earliest development of atherosclerosis. The present findings certainly underscore the need for well-designed, large prospective studies, aimed at evaluating the value of FMD as a bioassay in predicting cardiovascular events. Studies with thousands of subjects, such as the ongoing FATE trial,17 are underway to answer this question.

Over the last decade or so, the concept that vascular inflammation is the central orchestrator of atherosclerotic lesion formation, progression, and eventual rupture has emerged.26 Accordingly, there has been increased interest in evaluating inflammatory markers of atherosclerosis, of which CRP has emerged as one of the most important predictors of myocardial infarction, stroke, and vascular death in a variety of settings.27 CRP adds prognostic value to lipid screening, the metabolic syndrome and to the Framingham risk score, and only weakly correlates with the individual components of the Framingham risk score.11 Indeed, in the present study, correlation with individual risk factors was not particularly robust. In agreement with the recent study by Albert et al., the Framingham risk score was the best predictor of CRP variability with moderate correlation (r=0.36) in this cohort of men. These data suggest that CRP offers added value to conventional means of risk prediction.

Recent studies, including work from our laboratory, suggest that CRP is not only a predictor of atherosclerosis but also an active mediator in atherogenesis. CRP, at concentrations known to predict vascular disease has a direct effect to stimulate diverse early atherosclerotic processes including endothelial cell adhesion molecules, chemoattractant chemokines, and macrophage LDL uptake.20 In addition, CRP downregulates nitric oxide (NO) synthase-derived NO, while augmenting the production of the potent endothelium-derived vasoconstrictor endothelin -1.

Given the importance of both endothelial function and CRP in the development of atherosclerosis and the effects that CRP has on NO biology, one would predict that elevated CRP levels would correlate strongly with endothelial dysfunction.6 Therefore, it is somewhat surprising to learn that CRP is not related to endothelial function in this large cohort of otherwise healthy volunteers. This is in agreement with some but not all studies that have addressed this relationship in healthy individuals.9,28 Previous studies that have addressed this question have suffered from limited sample size. Schindler et al., demonstrated that in patients with a normal coronary angiogram, a correlation between endothelial function and elevated CRP levels was limited to those with advanced impairment of coronary vasoreactivity.29 Likewise, we found no correlation between FMD and CRP in the entire cohort, although a weak correlation was observed in a subgroup of patients with severe endothelial dysfunction and in active smokers. Yet, these subgroups correlations should be viewed as too weak to be of major diagnostic or prognostic significance.

Limitations
The results of the current study are only applicable to a relatively healthy cohort of men without co-existing vascular disease. However, this is a population of particular interest with respect to improving primary prevention risk detection and treatment.

While both CRP and FMD have been promoted as new biomarkers that may be predictive of coronary heart disease risk, the current study found no association between two biomarkers. Therefore, it remains to be determined whether performing brachial artery FMD testing may provide additional risk assessment over CRP in order to identify individuals at increased risk who might benefit from more aggressive primary prevention therapy.


    Acknowledgments
 
We thank all of the FATE coordinators and technicians from Calgary, Halifax, Hamilton and Montreal sites for their invaluable contributions. Special acknowledgements are extended to Dr. Erik Larsen, Beverly Madden, Shirley White, Shirley Jorge, Lyne Sattlegger and the staff of the Calgary Laboratory Services. This work was supported in part by grants from Pfizer, the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada to Dr. Todd J. Anderson.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: emerging mechanistic paradigms Circulation 1999;100:e20-e28.[Abstract/Free Full Text]
  2. Ridker PM, Cushman M, Stampfer MJ, et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men N. Engl. J. Med. 1997;336:973-979.[Abstract/Free Full Text]
  3. Ridker PM, Rifai N, Clearfield M, et al. Measurement of C-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events N. Engl. J. Med. 2001;344:1959-1965.[Abstract/Free Full Text]
  4. Anderson TJ, Uehata A, Gerhard MD, et al. Close relation of endothelial function in the human coronary and peripheral circulations J. Am. Coll. Cardiol. 1995;26:1235-1241.[CrossRef][Medline]
  5. Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis Lancet 1992;340:1111-1115.[Medline]
  6. Verma S, Wang CH, Li SH, et al. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis Circulation 2002;106:913-919.[Abstract/Free Full Text]
  7. Wang CH, Li SH, Weisel RD, et al. C-reactive protein upregulates angiotensin type 1 receptors in vascular smooth muscle Circulation 2003;107:1783-1790.[Abstract/Free Full Text]
  8. Jartti L, Raitakari OT, Jarvisalo MJ, et al. Higher prevalence of Chlamydia pneumoniae seropositivity in Finnish twins compared with co-twins living in Sweden: relationships with markers of subclinical atherosclerosis Clin. Sci. (Lond.) 2003;105:303-313.[Medline]
  9. Jarvisalo MJ, Harmoinen A, Hakanen M, et al. Elevated serum C-reactive protein levels and early arterial changes in healthy children Arterioscler Thromb Vasc Biol 2002;22:1323-1328.[Abstract/Free Full Text]
  10. Fichtlscherer S, Rosenberger G, Walter DH, et al. Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease Circulation 2000;102:1000-1006.[Abstract/Free Full Text]
  11. Albert MA, Glynn RJ, Ridker PM. Plasma concentration of C-reactive protein and the calculated Framingham Coronary Heart Disease Risk Score Circulation 2003;108:161-165.[Abstract/Free Full Text]
  12. Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events N. Engl. J. Med. 2002;347:1557-1565.[Abstract/Free Full Text]
  13. Vita JA, Treasure CB, Nabel EG, et al. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease Circulation 1990;81:491-497.[Abstract]
  14. Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease Circulation 2000;101:1899-1906.[Abstract/Free Full Text]
  15. Suwaidi JA, Hamasaki S, Higano ST, et al. Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction Circulation 2000;101:948-954.[Abstract/Free Full Text]
  16. Neunteufl T, Heher S, Katzenschlager R, et al. Late prognostic value of flow-mediated dilation in the brachial artery of patients with chest pain Am J Cardiol 2000;86:207-210.[CrossRef][Medline]
  17. Anderson TJ, Roberts AC, Hildebrand K, et al. The fate of endothelial function testing: rationale and design of the Firefighters And Their Endothelium (FATE) study Can J Cardiol 2003;19:61-66.[Medline]
  18. Anderson TJ, Elstein E, Haber H, et al. Comparative study of ACE-inhibition, angiotensin II antagonism, and calcium channel blockade on flow-mediated vasodilation in patients with coronary disease (BANFF study) J. Am. Coll. Cardiol. 2000;35:60-66.[CrossRef][Medline]
  19. Grundy SM, Pasternak R, Greenland P, et al. Assessment of cardiovascular risk by use of multiple-risk-factor assessment equations: a statement for healthcare professionals from the American Heart Association and the American College of Cardiology Circulation 1999;100:1481-1492.[Free Full Text]
  20. Yeh ET, Willerson JT. Coming of age of C-reactive protein: using inflammation markers in cardiology Circulation 2003;107:370-371.[Free Full Text]
  21. Verma S, Anderson TJ. Fundamentals of endothelial function for the clinical cardiologist Circulation 2002;105:546-549.[Free Full Text]
  22. Widlansky ME, Gokce N, Keaney JF, et al. The clinical implications of endothelial dysfunction J. Am. Coll. Cardiol. 2003;42:1149-1160.[CrossRef][Medline]
  23. Gokce N, Keaney Jr. JF, Hunter LM, et al. Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study Circulation 2002;105:1567-1572.[Abstract/Free Full Text]
  24. Celermajer DS, Sorensen KE, Bull C, et al. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction J. Am. Coll. Cardiol. 1994;24:1468-1474.[Medline]
  25. Leeson P, Thorne S, Donald A, et al. Non-invasive measurement of endothelial function: effect on brachial artery dilatation of graded endothelial dependent and independent stimuli Heart 1997;78:22-27.[Abstract]
  26. Libby P. Inflammation in atherosclerosis Nature 2002;420:868-874.[CrossRef][Medline]
  27. Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detection and prevention Circulation 2003;107:363-369.[Free Full Text]
  28. Khairy P, Rinfret S, Tardif JC, et al. Absence of association between infectious agents and endothelial function in healthy young men Circulation 2003;107:1966-1971.[Abstract/Free Full Text]
  29. Schindler TH, Hornig B, Buser PT, et al. Prognostic value of abnormal vasoreactivity of epicardial coronary arteries to sympathetic stimulation in patients with normal coronary angiograms Arterioscler Thromb Vasc Biol 2003;23:495-501.[Abstract/Free Full Text]