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European Heart Journal Advance Access published online on January 9, 2007
European Heart Journal, doi:10.1093/eurheartj/ehl441
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© The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Inflammatory, haemostatic, and rheological markers for incident peripheral arterial disease: Edinburgh Artery Study

Ioanna Tzoulaki1,*, Gordon D. Murray1, Amanda J. Lee2, Ann Rumley3, Gordon D.O. Lowe3 and F. Gerald R. Fowkes1

1 Wolfson Unit for Prevention of Peripheral Vascular Diseases, Public Health Sciences, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK
2 Department of General Practice and Primary Care, University of Aberdeen, Foresterhill Health Centre, Westburn Road, Aberdeen AB25 2AY, UK
3 Division of Cardiovascular and Medical Sciences, University of Glasgow, Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK

Received 1 May 2006; revised 20 November 2006; accepted 23 November 2006.

* Corresponding author. Tel: +44 131 650 6983; fax: +44 131 650 6904. E-mail address: i.tzoulaki{at}sms.ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusion
 Supplementary material
 Acknowledgements
 References
 
Aims Recently, markers of inflammation, haemostasis, and blood rheology have received much attention as risk factors for coronary heart disease and stroke. However, their role in peripheral arterial disease (PAD) is not well established and some of them, including the pro-inflammatory cytokine interleukin-6 (IL-6), have not been examined before in prospective epidemiological studies.

Methods and results In the Edinburgh Artery Study, we studied the development of PAD in the general population and evaluated 17 potential blood markers as predictors of incident PAD. At baseline (1987), 1519 men and women free of PAD aged 55–74 were recruited. After 17 years, 208 subjects had developed symptomatic PAD. In analysis adjusted for cardiovascular risk factors and baseline cardiovascular disease (CVD), only C-reactive protein, fibrinogen, lipoprotein (a), and haematocrit [hazard ratio (95% CI) corresponding to an increase equal to the inter-tertile range 1.30 (1.08, 1.56), 1.16 (1.05, 1.17), 1.22 (1.04, 1.44), 1.22 (1.08, 1.38)] were significantly (P<0.01) associated with PAD. However, these markers provided very little prognostic information for incident PAD to that obtained by cardiovascular risk factors and the ankle brachial index. Other markers including IL-6, intracellular adhesion molecule 1, D-dimer, tissue plasminogen activator antigen, and plasma and blood viscosities showed weak associations, which were considerably attenuated when CVD risk factors were accounted for.

Conclusions Our prospective data showed that several inflammatory, haemostatic, and rheological markers are associated with incident PAD; however, their clinical utility is likely to be limited. Future research is necessary to validate the importance of these biomarkers explicitly on PAD and to address the causality of the reported associations.

Key Words: Epidemiology • Peripheral arterial disease • Risk factors • Inflammation • Coagulation • Fibrinolysis • Blood viscosity


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusion
 Supplementary material
 Acknowledgements
 References
 
Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis and is associated with increased risk of cardiovascular morbidity and mortality.1,2 Large epidemiological studies have shown significant associations between PAD and major cardiovascular disease (CVD) risk factors including age, cigarette smoking, elevated blood lipids, diabetes, hypertension, obesity, and pre-existing CVD.3,4

However, as in coronary heart disease, many patients develop PAD in the absence of these factors. With recent advances in vascular biology, a number of biomarkers that represent activated inflammation and haemostasis and increased blood viscosity have been proposed as novel risk factors for CVD. Consequently, many epidemiological studies have evaluated their additive value to traditional CVD risk factors and have investigated their pathophysiological role in atherosclerotic development.5,6 However, few studies have examined the importance of these risk factors explicitly in PAD.7–11 For some biomarkers, including the pro-inflammatory cytokine interleukin-6 (IL-6), there are no prospective data on their association with the development of PAD.

This analysis used data collected over 17 years of follow-up from the Edinburgh Artery Study population based cohort established in 1987. We aimed to evaluate a series of plasma biomarkers as novel risk factors for the development of symptomatic PAD, and secondly to compare their effect with conventional risk factors for PAD. In addition, we sought to determine whether these markers had any incremental value above and beyond the information yielded by traditional risk factors and the ankle brachial index (ABI), a non-invasive marker of asymptomatic atherosclerosis. Biomarkers included in the analysis were markers of inflammation, such as C-reactive protein and IL-6, and adhesion molecules, such as intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and E-selectin. Also, we studied the association of lipoprotein (a) [Lp(a)] and several markers of activated coagulation and fibrinolysis, such as fibrinogen, D-dimer, tissue plasminogen activator antigen (t-PA), vonWillebrand factor (vWF), factor VII, fibrinopeptide A (FpA), and prothrombin fragment 1+2 (F1+2). Finally, we investigated the association of rheological markers, namely haematocrit and blood and plasma viscosities, with incident PAD.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusion
 Supplementary material
 Acknowledgements
 References
 
Study population
The Edinburgh Artery Study began in 1987 as a cross-sectional survey of 809 men and 783 women aged 55–74 and was designed to study the risk factors for PAD. This population, which was almost exclusively of white ethnicity, was selected at random, in 5-year age bands, from 11 general practices serving a range of socio-economic and geographic areas throughout the city. The response rate was 65%, and follow-up of a sample of non-responders showed no substantial bias. Details of the study recruitment and examination process have been described.12 Ethics Committee approval was given for this study, and informed consent was obtained from each subject. Subjects completed a self-administered questionnaire at baseline that contained validated questions regarding personal characteristics, social class, intermittent claudication and angina [World Health Organization (WHO) questionnaire],13 medical history, smoking, diet and physical activity.

Clinical examination
Subjects were invited for a comprehensive clinical examination at baseline. Clinical measurements were conducted by trained research staff during each examination. A 12-lead electrocardiogram (ECG) was taken and coded independently by two observers using the Minnesota code.14 Standing height (without shoes) was measured to the nearest 5 mm using a free standing metal ruler on a heavy base. Weight (without shoes and outer clothing) was measured to the nearest 100 g on digital scales (Soehnle). Body mass index (BMI) was calculated as the weight in kilograms divided by square of the height in metres. Systolic and diastolic (phase V) blood pressures were recorded in the right arm only, after 10 min rest in the supine position, using a stethoscope. Ankle systolic pressures were measured first in the right leg and then in the left leg at the posterior tibial artery, using a Sonicaid Doppler ultrasound probe and a random zero sphygmomanometer as previously described.15 The ABI was calculated by dividing the ankle systolic pressure by the brachial systolic pressure. The lower of the two leg indices was used in the analysis as indicative of worse disease. In the current analyses, three subjects at baseline had values of ABI above 1.50 and were excluded due to probable arterial rigidity.

Measurement of biochemical variables
A fasting 20 mL sample of venous blood and a urine sample were taken for estimation of biochemical, inflammatory, and haemostatic factors. All blood samples were centrifuged within 2 h of collection and were stored the same day at –40°C. Serum total cholesterol, high density lipoprotein (HDL) cholesterol, and blood glucose were performed on a Cobas Bio analyzer (Roche Products) using standard kits. The total/ HDL cholesterol ratio was calculated. Diabetic status of the subject was assessed in a number of ways. At baseline examination, a blood sample was taken for measurement of blood glucose and then each subject not known to be diabetic consumed 75 g of glucose in the form of 335 mL Solripe Gluctoza Health Drink (Strathmore Mineral Water Company, Forfar, Scotland). A second blood glucose specimen was taken 2 h after the oral glucose load. In addition, at baseline and 12-year examinations, self-reported diabetic status and use of insulin injections and tablets for diabetes were recorded. Subjects were classified as suffering from diabetes if (i) they had been told by a doctor that they suffered from diabetes and were receiving treatment or (ii) the glucose concentration of the 2 h blood sample was ≥11.1 mmol/L.

C-reactive protein was measured immunologically using a high-sensitivity assay in a BN ProSpec nephelometer (Dade Behring, Milton Keynes, UK). Plasma levels of IL-6, ICAM-1, VCAM-1, E-selectin, vWF, D-dimer, t-PA, and Lp(a) were measured using high sensitivity ELISA kits as previously described.8,15,16 Fibrinogen was measured in citrated plasma by a thrombin-clotting turbidometric method in a centrifugal analyser.17 Urinary FpA and leukocyte elastase were measured by RIA,18 plasma factor VII was measured by a functional antigenic assay, and plasma F1+2 was measured by ELISA (Dade Behring, Marburg, Germany) as previously described.11 Finally, blood and plasma viscosities were measured in a Coulter–Harkness viscometer at 37°C. Haematocrit was measured with a Hawksley microcentrifuge and a reader. Missing values in these markers were due to decreasing availability of plasma samples and were considered ‘data missing at random’. Internal quality control plasma was run with the assay for each marker. Also, measurements of markers that fell outside the 95% normal range were repeated.

Identification and coding of peripheral arterial events
To obtain details on PAD events, information was sought from the following sources: general practitioners, the Information and Statistics Division of the Scottish Executive, and the participants themselves (by annual questionnaire). All events were further investigated by using hospital or general practioner records to ensure that the protocol criteria were fulfilled. To identify deaths, each participant's record was flagged at the United Kingdom National Health Service Central Registry.

People suffering from PAD were subsequently divided into those with moderate PAD and those with severe PAD. Moderate PAD included people with intermittent claudication (IC) only. Severe PAD included subjects who developed critical limb ischaemia (CLI) (rest pain, gangrene, or ulceration) or subjects who underwent surgical intervention (amputation or vascular surgery).

Baseline CVD was defined as myocardial infarction (MI) (two out of the three of subjects recall of doctor's diagnosis, positive WHO questionnaire, and ECG ischaemia), stroke, (recall of doctor's diagnosis), or angina (positive WHO questionnaire and either ECG ischaemia or recall of doctor's diagnosis).

Data analysis
Distributions of C-reactive protein, IL-6, ICAM-1, VCAM-1, E-selectin, leukocyte elastase, Lp(a), D-dimer, vWF, FpA, and F1+2 were positively skewed and were logarithmically transformed to approach normality. Similarly, t-PA and factor VII were square-root transformed. Ninety-eight subjects with C-reactive protein levels above 10 mg/l and 11 subjects with IL-6 levels above 100pg/mL were excluded from all analyses because these levels indicate the presence of acute inflammatory disease. Pack-years of smoking were calculated as years of cigarette smoking multiplied by the average number of packs smoked per day with the value zero entered for lifelong non-smokers. The distribution of pack-years was skewed and a square-root transformation was used in all analyses. Physical activity was used as a categorical variable with four groups comprising no activity, light activity, moderate activity, and strenuous activity. Hypertension was defined as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg or use of drugs to lower blood pressure at baseline.

To compare mean levels of risk factors at baseline in subjects who developed PAD and who did not, the independent sample t-test was used for continuous variables and the {chi}2 test for categorical variables. A test for trend across disease categories and baseline risk factors was performed using ANOVA.

All the subjects of the Edinburgh Artery Study were recruited at baseline over a period of 1 year and therefore follow-up time is essentially constant. Duration of follow-up was calculated in person-years by using the follow-up of each participant from the baseline examination until death, PAD event, or most recent follow-up assessment. Cox proportional hazard models were used to estimate risk of PAD. If more than one event was recorded (for example, both intermittent claudication and CLI), the first event was used for the calculation of the disease-free survival time. Hazard ratios (95% CI) for BMI, diabetes, total/ HDL cholesterol ratio, hypertension, pack-years of smoking, physical activity, and CVD (MI, stroke, or angina) at baseline adjusted for age and sex and then for all variables were calculated with Cox regression. These variables were selected because they are considered as established risk factors for PAD and also because they are potential confounders due to their associations with markers studied here.

Next, we calculated the hazard ratio for each inflammatory, haemostatic, and rheological variable adjusted for age and sex (i) plus BMI, diabetes, total/HDL cholesterol ratio, pack-years smoking, physical activity, and hypertension and (ii) CVD at baseline (MI, stroke, or angina). In order to allow comparisons between the different hazard ratios, all inflammatory, haemostatic, and rheological variables were divided by the inter-tertile range (of the transformed distribution). Therefore, the hazard ratios are actually corresponding to an increase in the covariate equal to the inter-tertile range.

Receiver operator curves (ROC) were used to examine the additive predictive value of inflammatory, haemostatic, and rheological markers to traditional risk factors and ABI model in discriminating subjects into those who suffered or did not suffer from a PAD event during the follow-up. In this analysis, hypertension was not added in the traditional risk factors model due to its high correlation with the ABI.

The proportional hazards assumption of invariant hazard ratio was visually tested (by plotting log–log survival plots) and found to be satisfactory for all models constructed. Throughout all analyses, a two-sided P≤0.05 was taken to denote statistical significance. All analyses were performed using SPSS v13 for Windows.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusion
 Supplementary material
 Acknowledgements
 References
 
At baseline, 1519 subjects (51% males) were free of PAD with mean (SD) age 67.7 (5.7) years. During the subsequent 17 years, 209 (14%) developed symptomatic PAD. Of those, 169 (81%) were diagnosed with moderate PAD (IC only). Also, 40 (19%) were diagnosed with severe PAD since they experienced the more severe symptoms of CLI (rest pain, gangrene, or ulcer) or underwent intervention (amputation or vascular surgery). Among the 40 with severe PAD, 26 (65%) were also diagnosed with IC. Finally, during the follow-up period, 702 participants (46.2% of the baseline population) died, including 194 (27.6%) due to MI or stroke.

Traditional risk factors
Mean levels of plasma total cholesterol, ratio of total/ HDL cholesterol, cigarette smoking, and systolic blood pressure and prevalence of hypertension and baseline CVD were all significantly higher at baseline among subjects who subsequently developed PAD (Table 1). Conversely, levels of HDL cholesterol were significantly lower in those who developed PAD. Relatively more subjects with PAD had diabetes but the difference from the healthy group did not reach statistical significance. No significant differences were found for BMI, diastolic blood pressure, and physical activity (Table 1).


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  		Table 1 Mean (SD) and percentages of cardiovascular risk factors in 1519 subjects at baseline by development of future PAD

 
Table 2 presents age and sex-adjusted and multivariable adjusted hazard ratios (95% CI) for development of PAD for total/HDL cholesterol, pack-years smoking, hypertension, diabetes, BMI, physical activity, and baseline CVD. In the multivariable analysis, total/HDL cholesterol ratio, hypertension, smoking, and baseline CVD were all significantly associated with incident PAD (P<0.001).


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  		Table 2 Hazard ratios (95% CI) of cardiovascular risk factors for development of PAD (n=1434)

 
Inflammatory, haemostatic, and rheological markers
Overall, levels of these plasma markers studied here were highly significantly and positively correlated (Supplementary material online). The highest correlations observed were those between fibrinogen, C-reactive protein, and IL-6 which all had correlation coefficients greater than 0.50 (P<0.001).

Inflammatory markers [C-reactive protein, IL-6, ICAM-1, Lp(a)], haemostatic factors (fibrinogen, D-dimer, t-PA), and all rheological markers were significantly elevated at baseline in those subjects who experienced symptomatic PAD during the follow-up period (Table 3). A significant trend between higher levels of ICAM-1 (P=0.02), D-dimer (P=0.01), t-PA (P=0.02), and haematocrit (P<0.001) and worsening disease categories from no disease to moderate PAD (IC only) through to severe PAD (CLI or surgical intervention) was observed (Figure 1). Other markers did not show a clear trend across moderate and severe PAD.


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  		Table 3 Median (25th, 75th quartiles) of inflammatory, haemostatic, and rheological markers measured at baseline

 

Figure 4411
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  		Figure 1 Median (25th, 75th quartiles) of ICAM-1, D-dimer, t-PA, and haematocrit in people who remained free of PAD and in those who developed moderate PAD (intermittent claudication) and severe PAD (CLI or surgical intervention) after 17 years. The vertical bars denote those values of each marker that lie within 1.5 inter-quartile ranges of the limit of the inter-quartile range. The P-value for linear trend was 0.02 for ICAM-1, 0.01 for D-dimer, 0.02 for t-PA, and <0.001 for haematocrit.

 
Hazard ratios (95% CI) for PAD corresponding to an increase equal to the inter-tertile range of each marker with various degrees of adjustment are shown for all markers in Table 4. C-reactive protein had marginally the greatest hazard ratio compared with other markers [1.30 (95% CI: 1.08, 1.56)] and retained significance in the multivariate model. ICAM-1 and IL-6 showed significant associations in analyses adjusted for age and sex. However, their hazard ratios were attenuated after adjustments for cardiovascular risk factors and then for CVD at baseline. In the fully adjusted analysis, their hazard ratios lost statistical significance. For example, the hazard ratio (95% CI) of IL-6 was reduced from 1.21 (1.06, 1.39) to 1.06 (0.91, 1.23) after adjustments for risk factors and baseline CVD. In contrast, Lp(a) was little affected by cardiovascular risk factors and displayed significant associations even in the multivariable analysis. Leukocyte elastase, VCAM-1, and E-selectin did not show any significant associations with incident PAD.


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  		Table 4 Hazard ratios (95% CI) for development of PAD (n 924–1420)

 
Among the haemostatic markers, fibrinogen remained significantly associated with incident PAD independent of conventional risk factors and CVD at baseline, and its multivariate hazard ratio was 1.16 (1.05, 1.17) (Table 4). Levels of D-dimer and t-PA were also significantly associated in age and sex-adjusted analysis only [hazard ratio corresponding to an increase equal to the inter-tertile range (95% CI) 1.12 (1.01, 1.26) and 1.27 (1.12, 1.44), respectively], whereas no evidence for associations with incident PAD was seen for vWF, factor VII, FpA, or F1+2 (Table 4).

Finally, increased haematocrit showed significant associations with PAD when adjusted for risk factors and baseline CVD (Table 4). When we further adjusted haematocrit for fibrinogen, a major determinant of viscosity, associations were essentially unchanged and both fibrinogen and haematocrit retained significant association in the final model.

Area under the ROC analysis
The ABI is considered a powerful predictor of future symptomatic PAD. The incremental benefit of considering inflammatory, haemostatic, or rheological markers in addition to conventional risk factors and ABI to discriminate incident symptomatic PAD cases was also investigated by calculating the area under the ROC (AUROC) curve. The AUROC (95% CI) of the core model including conventional risk factors (age, sex, diabetes, total/ HDL cholesterol, smoking, BMI, physical activity, and history of CVD) and the ABI was 76.2% (71.1, 81.3). When individual markers were entered in the model, the AUROC was little increased. The highest increase was observed after fitting C-reactive protein [AUROC (95%) CI: 77.0% (72.1, 82.0)] or fibrinogen (77.4% (73.6, 81.2)). Moreover, even in models with cardiovascular risk factors alone (without ABI), inflammatory, haemostatic, or rheological markers did not manage to add considerably the discriminating power of the model. An example in the AUROC curve for different models with conventional risk factors, ABI, and C-reactive protein is presented in Table 5.


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  		Table 5 AUROC for prediction models with cardiovascular risk factors with and without ABI and C-reactive protein

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusion
 Supplementary material
 Acknowledgements
 References
 
Approximately 14% of the baseline population developed symptomatic PAD defined as IC, CLI, or surgical intervention after 17 years of follow-up. The main finding of this study was that elevated levels of C-reactive protein, fibrinogen, Lp(a), and haematocrit showed strong and independent associations with incident PAD. In contrast, IL-6, ICAM-1, D-dimer, t-PA, plasma, and blood viscosity showed weaker associations, which were considerably attenuated when cardiovascular risk factors were accounted for. Most importantly, these markers added little prognostic information to traditional risk factors and ABI for risk of development of PAD.

Traditional risk factors
Our prospective data confirm the importance of major CVD risk factors in PAD development. Smoking, total/HDL cholesterol, hypertension, and pre-existing CVD were strong independent predictors of PAD development as previously described.3,4 Despite the high prevalence of diabetes in the group of people who developed PAD, it did not reach statistical significance probably due to low statistical power. Physical activity has not been extensively investigated in relation to PAD development. Here, we did not find any clear associations between decreased physical activity and PAD development.

Inflammatory markers
IL-6 and C-reactive protein have received much attention recently for their role in atherosclerotic development.19 IL-6 is thought to contribute to atherosclerosis through various mechanisms since it up-regulates C-reactive protein, fibrinogen, and other acute phase reactants, enhances endothelial cell adhesiveness, activates the production of tissue factor and vWF, decreases anticoagulant levels, and leads to more thrombogenic platelets.20 In addition, increased C-reactive protein itself has pro-inflammatory properties including up-regulation of adhesion molecule expression, activation of complement, and mediation of LDL uptake by macrophages.21

IL-6 has shown independent associations in several epidemiological studies on coronary heart disease and stroke in various populations.19 In the present analysis, increased IL-6 levels were associated with PAD risk, but after adjustment for cardiovascular risk factors these associations were considerably attenuated. This is the first study to assess the role of IL-6 in relation to PAD prospectively and therefore our results need cautious interpretation. They may reflect a modest effect of IL-6 on PAD and therefore a possible difference in its predictive ability across diverse disease manifestations. Nonetheless, IL-6 was measured at baseline in fewer individuals than other markers studied here and this might have itself somewhat influenced the precision of the estimated effect sizes and the significance levels.

C-reactive protein has been extensively studied as a cardiovascular risk factor; yet, only one prospective study on PAD has been reported.9 In accordance, we reported here a significant hazard ratio for C-reactive protein and incident PAD in analysis adjusted for conventional risk factors and history of CVD at baseline. However, concern has been recently raised on the clinical utility of C-reactive protein in clinical risk prediction which cannot be judged by the significance and independence of the reported hazard ratios.22 In the Edinburgh Artery Study, we assessed the clinical utility of C-reactive protein over and above cardiovascular risk factors and the ABI, a useful screening tool for PAD. Interestingly, C-reactive protein levels added very little prognostic information to traditional cardiovascular risk factors and ABI, and thus are not likely to contribute to the assessment of peripheral risk.

Adhesion molecules play a role in early leukocyte adhesion in the vessel wall and in the firm attachment of leukocytes and their emigration into the arterial wall. Studies on their role in PAD are also limited. Here, in agreement with a previous report,7 levels of ICAM-1, but not VCAM-1, were associated with incident PAD; however, they did not retain statistical significance after adjustment for traditional risk factors. No evidence was found for an association of E-selectin and PAD development which is in agreement with previously published negative results on association with the presence and progression of peripheral atherosclerosis.16,23,24

To the best of our knowledge, this is the first study to examine leukocyte elastase, a marker of leukocyte activation, with future PAD development and no evidence for a significant association was found. In contrast, Lp(a) was independently associated with development of PAD, in support of previous studies on PAD25–27 and a meta-analysis on coronary heart disease.28 Nevertheless, the pathological role of Lp(a) in atherogenesis remains unknown, although it has been proposed that Lp(a) acts as a pro-inflammatory mediator for lesion formation and up-regulates adhesion molecule expression and inhibits fibrinolytic activity by inducing the production of plasminogen activator-1.29

Haemostatic markers
Our findings agree with previously published generally negative results on coronary heart disease and on PAD for factor VII, F1+2, and FpA.11,30,31 vWF has shown modest associations with coronary heart disease in recent meta-analysis.32 Here, in accordance with previous results on PAD development11 as well as on asymptomatic PAD,30,31 we found no evidence for association of vWF with incident PAD.

Levels of t-PA and D-dimer reflect plasmin and fibrin formation, respectively. Both markers have been extensively studied in relation to CHD; however, epidemiological data on PAD are scarce.33,34 In previous analysis of the Edinburgh Artery Study, t-PA and D-dimer were not associated with incident PAD after 5 years of follow-up.11 However, both plasma markers have shown higher levels with the presence and increasing severity of PAD in cross-sectional analyses.35,36 Here, both markers were associated with PAD in age and sex-adjusted analysis but not in the multivariable model. Nevertheless, they showed a dose–response relationship with disease severity which further supports a role of these molecules in peripheral atherosclerosis.

Our results also highlight a strong and independent association between increased fibrinogen levels and PAD development. Fibrinogen is an acute phase reactant, a major determinant of blood viscosity, and a marker of activated coagulation and may be involved in early and late stages of atherosclerotic development through multiple mechanisms.37 Over the past 20 years, it has shown compelling evidence for an independent association with coronary heart disease, stroke, PAD, and asymptomatic atherosclerosis.38 An individual patient meta-analysis has recently demonstrated strong associations between fibrinogen and vascular and non-vascular mortality.39 Nevertheless, in the present analysis, the incremental benefit of fibrinogen over and above traditional risk factors and ABI for PAD assessment was very limited. Thus, like C-reactive protein, fibrinogen might have a role in PAD, but its value as a useful tool for clinical risk prediction is open to doubt.

Blood rheology
Markers of blood viscosity have previously been shown to be associated with PAD and with coronary heart disease.40 Our prospective data indicate that their effect on PAD was modest and that it was considerably reduced after adjustment for conventional risk factors. Nevertheless, increased haematocrit levels retained significant associations with PAD independent of cardiovascular risk factors and of fibrinogen and also presented a dose–response relationship with disease severity. However, as other markers studied here, it is not likely to have clinical value in PAD risk prediction.

Limitations
Most studies on PAD have defined the disease according to the presence of symptoms of intermittent claudication only. In the present study, we included the more severe events of CLI and surgical interventions to increase the generalizability of our findings. However, we did not include asymptomatic cases assessed by the ABI because this measurement was not available after 17 years of follow-up. Therefore, the incidence of PAD is probably underestimated. A number of other limitations of this study need to be considered. First, the generalizability of our results to other ethnic groups and ages is unknown. Also, all the biochemical markers were measured only once and intra-individual variation could not thus be taken into account. However, this would tend to result in an underestimation of the true effect. Furthermore, we did not adjust our analysis for aspirin or statin use at baseline, but at that time (1987/88) very few of the Edinburgh population took aspirin for the prevention of CVD, and statins had not been introduced.


   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
Supplementary material
 Acknowledgements
 References
 
In this prospective study, we confirmed the importance of conventional cardiovascular risk factors on PAD and also we demonstrated that several markers of inflammation, haemostasis, and rheology were independently associated with incident PAD and its severity. C-reactive protein, fibrinogen, Lp(a), and haematocrit showed strong associations which were independent of cardiovascular risk factors. However, their use in the assessment of individual PAD risk is likely to be limited.


   Supplementary material
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Supplementary material
Acknowledgements
 References
 
Supplementary material is available at European Heart Journal online.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Supplementary material
 Acknowledgements
References
 
We acknowledge the financial support from the British Heart Foundation. We thank Professor Joan Dawes for assay of urinary FpA and all participants, staff, and general practitioners involved in the Edinburgh Artery Study. I.T. is funded by the Scottish Executive (Chief Scientist Office).

Conflict of interest: none declared.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Supplementary material
 Acknowledgements
 References
 

  1. Leng GC, Lee AJ, Fowkes FGR, Whiteman M, Dunbar J, Housley E, Vaughan Ruckley C. (1996) Incidence, natural history and cardiovascular events in symptomatic and asymptomatic peripheral arterial disease in the general population. Int J Epidemiol 25:1172–1181.[Abstract/Free Full Text]
  2. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ, Browner D. (1992) Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med 326:381–386.[Abstract]
  3. Murabito JM, D'Agostino RB, Silbershatz H, Wilson WF. (1997) Intermittent claudication. A risk profile from The Framingham Heart Study. Circulation 96:44–49.
  4. Fowkes FG, Housley E, Riemersma RA, Macintyre CC, Cawood EH, Prescott RJ, Ruckley CV. (1992) Smoking, lipids, glucose intolerance, and blood pressure as risk factors for peripheral atherosclerosis compared with ischaemic heart disease in the Edinburgh Artery Study. Am J Epidemiol 135:331–340.[Abstract/Free Full Text]
  5. Lowe GD, Rumley A, Whincup PH, Danesh J. (2002) Hemostatic and rheological variables and risk of cardiovascular disease. Semin Vasc Med 2:429–439.[CrossRef][Medline]
  6. Lowe GD. (2005) Circulating inflammatory markers and risks of cardiovascular and non-cardiovascular disease. J Thromb Haemost 3:1618–1627.[CrossRef][Web of Science][Medline]
  7. Pradhan AD, Rifai N, Ridker PM. (2002) Soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, and the development of symptomatic peripheral arterial disease in men. Circulation 106:820–825.
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