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).
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Abstract |
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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.
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Introduction |
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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.1416 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.
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Methods |
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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 (HewlettPackard) 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, 612 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, 13, <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.
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Results |
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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).
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Discussion |
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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 n3 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.
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Acknowledgments |
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References |
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