Majda Joras
email: majda_joras@yahoo.com

Pavel Poredoš

The association of acute exercise-induced ischaemia with systemic vasodilator function in patients with peripheral arterial disease

Keywords: exercise, flow-mediated vasodilation, ischaemia, peripheral arterial disease, vasodilator function

Patients with peripheral arterial disease (PAD) frequently experience ischaemic attacks of the affected tissues during exercise. The present study assesses the association of transient exercise-induced leg ischaemia with vasodilator function of the clinically unaffected brachial artery over the course of 4 hours. Thirty male patients with symptomatic PAD and 14 age-and sex-matched healthy controls were included in the study. They performed a treadmill exercise until intolerable exerciseinduced ischaemic pain occurred in the affected lower extremity, or for at most 10 min. Flow-mediated dilation (FMD) of the brachial artery was measured at baseline, 30 minutes, 2 hours and 4 hours after exercise. Baseline FMD values were significantly diminished in patients (7.03 ± 1.99% vs 8.22 ± 1.60% in controls, p = 0.009). A significant decrease in FMD was observed in patients after exercise (at 30 minutes: 3.92 ± 1.78% vs 7.03 ± 1.99% at baseline, p < 0.001; at 2 hours: 6.36 ± 2.12% vs 7.03 ± 1.99% at baseline, p = 0.005), followed by a gradual return to its baseline value, whereas FMD in controls non-significantly increased after exercise. The difference in the pattern of FMD change over time between patients and controls was significant (p < 0.001). This study shows that in PAD patients ischaemia during intermittent claudication is related to a transitory functional deterioration of the distant arteries. This indicates the harmful systemic effects of repeated ischaemic attacks during exercise and might explain the generalized and advanced nature of atherosclerotic disease in PAD patients.

Introduction

Many patients with peripheral arterial disease (PAD) experience ischaemic attacks in hypoperfused tissues during physical activityintermittent claudication. In these patients, atherosclerotic disease has been observed to be more advanced and generalized than in healthy individuals of comparable ages. [START_REF] Ouriel | Peripheral arterial disease[END_REF][START_REF] Brevetti | Increased inflammatory status and higher prevalence of three-vessel coronary artery disease in patients with concomitant coronary and peripheral atherosclerosis[END_REF] But exact mechanisms leading to enhanced atherosclerosis in PAD patients have not been well explained. Some studies have shown increased levels of pro-inflammatory cytokines in the circulation and additional deterioration of systemic vasodilator function shortly after intermittent claudication in PAD patients. [START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFα), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF][START_REF] Silvestro | Effect of propionylcarnitine on changes in endothelial function and plasma levels of adhesion molecules induced by acute exercise in patients with intermittent claudication[END_REF] These findings support the concept of harmful mediators repeat-edly released from peripheral tissues to the circulation after each exercise-induced ischaemic episode, with the effects exceeding their initial local nature. On the contrary, however, other studies indicate the long-term beneficial effects of physical activity on systemic vasodilator function, [START_REF] Brendle | Effects of exercise rehabilitation on endothelial reactivity in older patients with peripheral arterial disease[END_REF] on reducing cardiovascular events and mortality in PAD patients [START_REF] Garg | Physical activity during daily life and mortality in patients with peripheral arterial disease[END_REF] and on improvement of the exercise capability of PAD patients. [START_REF] Gardner | Exercise rehabilitation programs for the treatment of claudication pain: a metaanalysis[END_REF] Impaired vascular function is considered to be a risk factor for the development of atherosclerosis and cardiovascular events. [START_REF] Gonzalez | Endothelial function, inflammation, and prognosis in cardiovascular disease[END_REF] It is present in all stages of PAD, is already in asymptomatic patients, and is related to the severity of the disease. [START_REF] Silvestro | Inflammatory status and endothelial function in asymptomatic and symptomatic peripheral arterial disease[END_REF] One of the most widely used and until now well-recognized methods of vascular (dys)function assessment, is measurement of the capability of endotheliumdependent dilation of arteries during the provocation of nitric oxide (NO) release. It is diminished when reduced amounts of vasodilatory substances are released from the functionally damaged endothelial cells, above all NO. [START_REF] Celermajer | Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction[END_REF][START_REF] Healy | Endothelial cell dysfunction: an emerging endocrinopathy linked to coronary disease[END_REF] The capability of vasodilation depends not only on endothelial function, but also on vascular smooth muscle function. However, in patients with atherosclerotic vascular disease and/or risk factors, endothelial function is particularly impaired, while vascular smooth muscle function is principally unaffected. [START_REF] Silvestro | Inflammatory status and endothelial function in asymptomatic and symptomatic peripheral arterial disease[END_REF][START_REF] Poredoš | Relation of blunted dilation of the brachial artery in insulin-dependent diabetes mellitus to microalbuminuria[END_REF] To date, there are no published data on the duration of changes and vasodilator function in PAD patients after intermittent claudication. Therefore, the aim of this study was to assess the time course of changes in vasodilator function of the clinically unaffected brachial artery after acute exerciseinduced leg ischaemia in PAD patients. Since vasodilatory response of the conduit peripheral arteries might be provoked by an increased blood flow, [START_REF] Celermajer | Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction[END_REF] vasodilator function was investigated during reactive hyperaemia in the brachial artery and the term flow-mediated vasodilation (FMD) is used hereafter in this study.

Materials and methods

Participants and study protocol

Thirty patients with intermittent claudication (all males, median age (interquartile range): 60.6 (54.9-62.6) years, range 44.5-65.4 years) and 14 age-and sex-matched healthy volunteers (age 57.8 (52.4-63.0) years, range 45.1-65.2 years) were included. Patients were recruited to the study from the Department for Vascular Medicine at the University Medical Centre in Ljubljana, after exclusion of symptomatic coronary artery disease and cerebrovascular disease in the past year. They met the clinical criteria for stage II of the Fontaine classification, [START_REF] Fontaine | Die chirurgische behandlung der peripheren durchblutungsstörungen[END_REF] with the symptoms lasting at least 6 months. Except for intermittent claudication, they had no other exercise-limiting factors. All had stable PAD (the absence of disease deterioration, according to their medical reports) and no acute vascular event in the last 3 months before inclusion in the study. One patient had a history of clinically manifest coronary artery disease in the past. Participants had not been exposed to cigarette smoke for at least 6 months. Exclusion criteria were: diabetes, heart failure, atrial fibrillation, renal or liver disease, or inflammatory disease of any aetiology. All patients were receiving acetylsalicylic acid (100 mg daily), and some of them additionally pentoxifylline (1200 mg daily) and/or statin (simvastatin or atorvastatin, different daily doses). None of the patients had received any antihypertensive/vasoactive or other anti-inflammatory medications for at least 14 days, or any invasive treatment in the last 6 months before the study. After the exclusion of contraindications, acetylsalicylic acid (100 mg daily) was temporarily introduced in healthy control participants a week before the examinations. At least 2 days before the examinations, the participants were not exposed to significant physical exercise. Examinations were performed in the morning, after a 12-hour overnight fast, including from caffeine. All participants gave their informed consent to take part in the study, approved by the Slovene National Ethics Committee.

After taking their medical history and a brief physical examination, body height and weight were measured and body mass index (BMI) calculated (weight [kg]/height 2 [m]). Systolic and diastolic blood pressures were recorded and ankle systolic pressure was measured in both legs with a Doppler ultrasound probe. The measurements of brachial and ankle systolic pressures were repeated immediately and 5 min after exercise. On each occasion, the ankle-brachial index (ABI) was calculated for both legs and the lower value used in further analysis.

Blood samples were taken at the beginning of the study, after a 20-min rest, from the left antecubital vein. Patients performed exercisea constant load treadmill test (12°slope, 3.2 km/h)also continuing after the onset of ischaemic pain, until the pain in the affected lower extremity became intolerable. Then the walking distance up to the onset of intermittent claudication and the maximal walking distance were calculated. Control participants exercised under the same protocol for 10 min. The measurements of FMD were performed before exercise and then 30 min, 2 h and 4 h after exercise.

Ultrasound examinations

In all participants, non-invasive ultrasound studies of vascular function were performed using the standard 10-MHz linear array transducer of a highresolution ultrasound system (ATL Ultrasound, Bothell, USA).

Investigation of vascular function was based on the technique described by Celermajer, et al. [START_REF] Celermajer | Noninvasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF] The right brachial artery diameter proximal to the antecubital fossa and blood flow velocity were measured in the longitudinal plane at rest and during reactive hyperaemia. Reactive hyperaemia was achieved by rapid release of a pneumatic pressure cuff placed around the forearm and inflated to a suprasystolic pressure for 5 min. Blood flow velocity was measured by a mid-artery pulsed Doppler signal at an angle of 68°to the vessel, obtained at rest and during reactive hyperaemia 15 s after forearm cuff deflation. Brachial artery flow for both study conditions was calculated from blood flow velocity and arterial diameter. Measurements of brachial artery diameter were performed at the end of diastole, which was determined by simultaneous monitoring of the electrocardiogram. Brachial artery diameter was measured by an independent observer, at a fixed distance from a selected anatomic marker. Each measurement was performed three times, recorded and then averaged. FMD was expressed as the change in the end diastolic brachial artery diameter during reactive hyperaemia 60 s after forearm cuff deflation compared to its baseline value.

Blood sampling and analyses

Blood was sampled from the left antecubital vein, using a tourniquet applied for 1 min or less. No blood additives were used for basic haematological and biochemical examinations. For the analysis of fibrinogen and oxidatively modified low-density lipoprotein (ox-LDL), blood was allowed to flow directly into a siliconized glass vacuum tube containing 0.13 mol l -1 trisodium citrate. Immediately after sampling, the blood was centrifuged for 30 min in a refrigerated centrifuge at 4°C and 2000 g. Platelet-poor plasma was transferred to small plastic vials, frozen in liquid nitrogen and stored at -70°C until analysed. For the assay of C-reactive protein (CRP), serum samples were frozen in liquid nitrogen and stored at -70°C until analysed.

Serum CRP levels were determined using an immunonephelometric method (N Latex High Sensitivity CRP™, Dade Behring GmbH, Liederbach, Germany). Fibrinogen was assessed by the Clauss clotting method (Multifibren* U, Dade Behring, Marburg, Germany). [START_REF] Clauss | Gerinnungsphysiologische Schnellmetode zur Bestimmung des Fibrinogens[END_REF] Blood glucose concentration and lipid profile were determined using routine enzymatic methods and LDL cholesterol was calculated using the Friedewald formula. [START_REF] Friedewald | Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge[END_REF] An enzymelinked immunosorbent assay was used for the measurement of plasma ox-LDL (Mercodia Oxidized LDL ELISA, Mercodia AB, Uppsala, Sweden).

Statistical analysis

The SPSS 15.0 statistical package for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. The distribution of variables was tested with the Kolmogorov-Smirnov test. Variables with a normal distribution were shown as means and standard deviations, and differences between the groups were tested with the Student's t-test for independent samples or analysed by one-way analysis of variance (ANOVA). Since patient and control groups were not comparable regarding brachial artery diameter, the brachial artery diameter was adjusted for in further analysis where appropriate. Differences in FMD between the groups were analysed by one-way analysis of covariance (ANCOVA), with brachial artery diameter as a covariate. Variables with a non-normal distribution were shown as medians with ranges between the first and the third quartiles, while differences between two groups were tested with the Mann-Whitney rank sum test. The relationships between two cate-gorical variables were tested with the chi-squared test. For correlation analysis, Pearson's correlation coefficient was calculated for variables with normal distribution, and Spearman's rank-correlation coefficient for variables with non-normal distribution. Repeated measures of FMD were analysed by two-way mixed ANCOVA, with time of measurement as a repeated measure, the presence of symptomatic PAD as a between-groups variable, and baseline brachial artery diameter as a covariate. Repeated measures of brachial artery diameter and flow were analysed by two-way mixed ANOVA, with time of measurement as a repeated measure and the presence of symptomatic PAD as a between-groups variable. A two-tailed value of p < 0.05 was considered statistically significant.

Results

Clinical and biochemical characteristics

Baseline characteristics of patients and controls are shown in Table 1. We found a past smoking history, hyperlipoproteinaemia and untreated mild or moderate arterial hypertension more frequent in patients than in controls. Out of 28 (93%) patients with hyperlipoproteinaemia, 23 (82%) were insufficiently treated.

All participants completed the study without any serious adverse events. Except for a longer period since cessation of smoking, higher ABI, and lower serum triglycerides and plasma ox-LDL in controls, there were no significant differences in clinical and biochemical characteristics between patients and controls (Table 2). Plasma ox-LDL was significantly higher in patients (55.8 ± 17.1 U l -1 vs 6.3 ± 1.6 U l -1 in controls, p < 0.001), and in patients was negatively correlated with time since smoking cessation (r = -0.41, p = 0.027).

In patients, the median walking distance up to the onset of intermittent claudication was 82 m (interquartile range 59-112 m), and the median value of maximal walking distance was 220 m (interquartile range 120-471 m). The leg with the lowest ABI was always the leg with the limiting claudication.

Of 30 patients included, 20 (67%) were receiving statins, and among the latter only 5 (25%) were achieving target lipid values according to NCEP ATP III Guidelines. Serum lipid values were lower in patients with statin treatment, most prominently LDL cholesterol (2.45 ± 0.73 mmol l -1 vs 3.16 ± 1.42 mmol l -1 in patients without statin treatment, p = 0.079). However, we found no significant differences in baseline clinical and biochemical characteristics between patients with or without statin treatment (data not shown).

Haemodynamic measurements

Before performing ultrasound measurements of vascular function, we investigated the morphology of the examined brachial artery segment. We found no disease-induced changes of the arterial wall structure and no indirect signs of the involvement of proximal brachial artery disease (e.g. pathological blood flow pattern) in any participant.

Values of resting brachial artery diameter were significantly lower in patients (4.41 ± 0.51 mm vs 4.80 ± 0.37 mm in controls, p = 0.015). But there was no significant difference in hyperaemic brachial artery flow between both the groups studied (303 ± 113 ml min -1 in patients vs 286 ± 99 ml min -1 in controls, p = 0.648). In comparison with healthy controls, patients had borderline diminished capa-bility of baseline FMD (7.03 ± 1.99% vs 8.22 ± 1.60%, p = 0.057). This difference was significant after adjustment for brachial artery diameter (p = 0.009). The baseline FMD value was higher in patients with statin treatment, but the difference between patients with or without statin treatment was not significant (7.24 ± 1.81% vs 6.60 ± 2.36% in patients without statin treatment, p = 0.413).

Vasodilator function after intermittent claudication

The values of resting brachial artery diameter, heart rate and blood pressure did not change significantly between the repeated FMD measurements. Hyperaemic brachial artery flow did not change significantly after exercise in patients, but it was significantly increased after exercise in controls (Table 3). However, the difference in hyperaemic brachial artery flow between both the groups studied was significant only 2 hours after exercise (276 ± 115 ml min -1 in patients vs 367 ± 124 ml min -1 in controls, p = 0.021). FMD decreased at 30 min after exercise and then gradually increased to its baseline value in patients, contrary to a slight non-significant increase after exercise in controls (Figure 1). The difference in FMD change between patients and controls over time was significant (F (1.86, 76. 3. In comparison with its baseline value, FMD was significantly decreased in patients at 30 min (p < 0.001) and at 2 h after exercise (p = 0.005). In comparison with controls, the FMD value was significantly lower in patients at 30 min (p < 0.001), 2 h (p < 0.001) and 4 h after exercise (p = 0.002).

Analysis of correlations between FMD and different parameters

The correlation coefficients between FMD values and different parameters in all participants are shown in Table 4. In all participants together, the baseline FMD was significantly negatively related to age (r = -0.41, p = 0.006) and arterial hypertension (r = -0.36, p = 0.017). We also found significant correlations between FMD values after exercise (at 30 min or 2 h) and the presence of clinically manifest PAD, arterial hypertension, ABI, maximal walking distance, baseline FMD and plasma ox-LDL. There were no significant correlations between FMD values and time since smoking cessation, serum lipids, blood glucose and CRP.

Discussion

The present study describes the association of acute ischaemia during intermittent claudication in PAD patients with systemic vasodilator function over the course of several hours. This issue is of importance in view of the existing interest in prescribing physical training as a crucial part of the treatment of patients with atherosclerotic vascular disease, [START_REF] Ignarro | Nutrition, physical activity, and cardiovascular disease: an update[END_REF] especially PAD. [START_REF] Gardner | Exercise rehabilitation programs for the treatment of claudication pain: a metaanalysis[END_REF][START_REF] Stewart | Exercise training for claudication[END_REF] There is evidence of the long-term beneficial effects of physical activity in PAD patients. [START_REF] Brendle | Effects of exercise rehabilitation on endothelial reactivity in older patients with peripheral arterial disease[END_REF][START_REF] Garg | Physical activity during daily life and mortality in patients with peripheral arterial disease[END_REF][START_REF] Stewart | Exercise training for claudication[END_REF] In contrast, some studies [START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFα), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF] have suggested that exercise-induced transitory leg ischaemia in these patients leads to a release of anaerobic metabolites and their products to the circulation during the reperfusion period, with potentially harmful systemic effects. However, the results of the latter studies could be biased by the influence of medications and concomitant symptomatic atherosclerotic vascular disease of other locations in some participants, as well as the essential differences in the expression of some atherosclerotic risk factors between patients and controls (current smoking status, diabetes). Impaired vascular function has been recognized as the earliest measurable change of the arterial wall in atherogenesis [START_REF] Celermajer | Noninvasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF] and a predictor of future cardiovascular events in patients with atherosclerotic vascular disease. [START_REF] Brevetti | Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease. Additive value of flowmediated dilation to ankle-brachial pressure index[END_REF] In the present study, we performed the widely used non-invasive ultrasound measurement of FMD [START_REF] Celermajer | Noninvasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF] for studying vasodilator function. It was derived from previous observations that FMD additionally decreased in PAD patients shortly after exercise-induced maximal tolerable leg ischaemia. [START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF][START_REF] Silvestro | Effect of propionylcarnitine on changes in endothelial function and plasma levels of adhesion molecules induced by acute exercise in patients with intermittent claudication[END_REF] To assess the duration of this phenomenon, we extended the observation time to 4 hours. Therefore, we followed the time course of changes in FMD of the clinically unaffected brachial artery after acute exercise-induced leg ischaemia in PAD patients.

As expected, we found significantly lower FMD values in PAD patients throughout the study. Our results further showed decreased FMD in PAD patients 30 min after exercise, followed by its gradual increase back to the baseline value at 4 hours after exercise, while in controls no significant changes of FMD were observed over time (Figure 1). A transitory decrease of FMD occurred in PAD patients after intermittent claudication in the absence of significant changes in hyperaemic flow stimulus and resting vessel diameter. This indicates the link between acute exercise-induced ischaemia and vasodilator function. The gradual improvement of FMD after cessation of ischaemic stimulus in PAD patients could be achieved through activation of adaptational mechanisms responsible for NO release and vascular function.

This study demonstrated non-significantly higher baseline FMD values in a subgroup of PAD patients with statin treatment. However, in view of the relatively small number of patients in each subgroup (with and without statin treatment), these results do not exclude a more impaired vasodilator function in patients without statin treatment, as indicated in recent studies. [START_REF] Jarvisalo | HMG CoA reductase inhibitors are related to improved systemic endothelial function in coronary artery disease[END_REF][START_REF] Taneva | Early effects on endothelial function of atorvastatin 40 mg twice daily and its withdrawal[END_REF] Also, this discrepancy between previous studies and the present results might partly be attributed to undertreatment with statins, leading to not entirely recognized statin effects in our PAD patients. Considering the NCEP ATP III Guidelines, [START_REF] Grundy | Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines[END_REF] the majority of PAD patients in the present study had insufficiently treated hyperlipoproteinaemia.

The baseline FMD values observed in our study in PAD patients were comparable with those in other studies performed in this clinical setting. [START_REF] Brevetti | Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease. Additive value of flowmediated dilation to ankle-brachial pressure index[END_REF][START_REF] Loffredo | Oxidativestress-mediated arterial dysfunction in patients with peripheral arterial disease[END_REF] On the contrary, baseline FMD values in the controls were lower than those observed in many other studies, [START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF][START_REF] Loffredo | Oxidativestress-mediated arterial dysfunction in patients with peripheral arterial disease[END_REF] although still significantly higher than in PAD patients.

The lower FMD values observed in controls in the present study could be explained by the presence of some atherosclerotic risk factors, particularly age (over 40 years). [START_REF] Celermajer | Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women[END_REF] Vascular dysfunction is considered as a forerunner of atherosclerotic presentations of cardiovascular disease, [START_REF] Gonzalez | Endothelial function, inflammation, and prognosis in cardiovascular disease[END_REF] preceding other measurable changes in atherogenesis. [START_REF] Poredoš | Endothelial dysfunction and cardiovascular disease[END_REF] As ABI probably has insufficient sensitivity for the detection of early atherosclerotic changes in the arterial wall, [START_REF] Mcdermott | Ankle-brachial index and subclinical cardiac and carotid disease. The multi-ethnic study of atherosclerosis[END_REF] it could be assumed that perhaps initial subclinical atherosclerosis was already present in these controls. This assumption is further supported by the lack of significant differences in CRP and fibrinogen levels between the PAD patients and controls in this study, as higher levels of both inflammatory markers were demonstrated in atherosclerotic vascular disease in previous studies. [START_REF] Brevetti | Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden[END_REF][START_REF] Ridker | Novel risk factors for systemic atherosclerosis. A comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease[END_REF] To avoid the possible influence of acetylsalicylic acid on inflammatory markers in this study, it was introduced temporarily in controls than run the risk of potential vascular events after its withdrawal in PAD patients. [START_REF] Blinc | Pharmacological prevention of atherothrombotic events in patients with peripheral arterial disease[END_REF] The expression of atherosclerotic risk factors was partially unbalanced in both examined groups. Arterial hypertension was more frequent in PAD patients than in controls, but further analysis revealed no significant differences in the absolute values of systolic and diastolic blood pressure between the two groups. However, we could not avoid the shorter time since smoking cessation and higher serum triglycerides in PAD patients.

A significantly higher plasma ox-LDL was demonstrated in PAD patients, associated with time since smoking cessation. This finding is in line with the results of a previous study that reported increased circulating ox-LDL in atherosclerotic vascular disease, also at the subclinical stage. [START_REF] Holvoet | for the Multi-Ethnic Study of Atherosclerosis. The relationship between oxidized LDL and other cardiovascular risk factors and subclinical CVD in different ethnic groups: The Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF] Furthermore, in patients with atherosclerotic risk factor hypercholesterolemia, plasma ox-LDL was found to be associated with vascular function. [START_REF] Matsuoka | Endothelial dysfunction associated with oxidative stress in human[END_REF] In general, plasma ox-LDL in the present study was associated with FMD values throughout the study, considering all individuals (patients and controls) investigated together. Since patients with coronary artery disease and higher oxidative stress and impaired vascular function were observed to be particularly prone to future cardiovascular events, [START_REF] Heitzer | Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease[END_REF] it can be presumed that the additional decrease in FMD associated with intermittent claudication, although transitory and of short duration, may be responsible for the progression of and the more advanced stage of atherosclerosis in PAD patients.

Study limitations

PAD patients with diabetes mellitus were not included in the study due to their expected (at least subclinical) neuropathy, [START_REF] Boulton | Diabetic neuropathies. A statement by the American diabetes association[END_REF] with consequent disturbance in the perception of pain and/or possible different vascular response. Furthermore, we omitted FMD measurement immediately after exercise, since the expected increase in baseline brachial artery diameter at that time might result in a decreased additional FMD response during reactive hyperaemia as a consequence of systemic vasodilation caused by exercise.

Conclusion

This study shows that in PAD patients ischaemia during intermittent claudication is related to a transitory decrease of FMD of the clinically unaffected distant brachial artery. These findings indicate that acute local exercise-induced ischaemia is associated with transitory generalized functional deterioration of the arterial wall. Repeated local ischaemic events during intermittent claudication, associated with potentially harmful systemic effects, might explain the more advanced and generalized nature of atherosclerotic disease in PAD patients.

  27) = 33.63, p < 0.001). Comparing each point in time after exercise with the baseline, we found significant differences in FMD change between patients and controls at 30 min (F (1, 41) = 57.68, p < 0.001), 2 h (F (1, 41) = 7.55, p = 0.009) and 4 h (F (1, 41) = 4.79, p = 0.034) after exercise. The detailed results of further analysis are shown in Figure 1 and Table

Figure 1

 1 Figure 1 Flow-mediated vasodilation (FMD) at baseline and after exercise (at 30 min, 2 h and 4 h) in patients (n = 30) and controls (n = 14). Data are presented as mean values, for clearer visualization without error bars. *p < 0.01 between patients and controls. p 1 , compared with baseline in patients.

Table 1

 1 Baseline characteristics of PAD patients and controlsData are presented as a number (percentage) or as a median value and interquartile range. The p-value refers to comparison of patients and controls by the Mann-Whitney rank-sum test or chi-squared test, where feasible. PAD, peripheral arterial disease.

	Parameter	Patients (n = 30)	Controls (n = 14)	p-value
	Age (years)	60.6 (54.9-62.6)	57.8 (52.4-63.0)	0.640
	Smoking history
	Current smokers	0	0
	Former smokers	29 (97%)	6 (43%)
	Time since smoking cessation (years)	2.0 (1.0-6.0)	15.5 (12.2-19.7)	< 0.001
	Diabetes	0	0
	Arterial hypertension	14 (47%)	3 (21%)	0.184
	Hyperlipoproteinaemia	28 (93%)	9 (64%)
	Medications
	Acetylsalicylic acid	30 (100%)	30 (100%)
	Pentoxifylline	6 (20%)	0
	Statin	20 (67%)	0

Data are presented as mean values ± SD or as median values and interquartile range. The p-value refers to comparison of patients and controls by the independent t-test or Mann-Whitney rank-sum test. PAD, peripheral arterial disease; ABI, ankle-brachial index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CRP, C-reactive protein; ox-LDL, oxidatively modified low-density lipoprotein.

Table 3

 3 Haemodynamic measurements at baseline and after exercise (at 30 min, 2 h and 4 h) in patients (n = 30) and controls (n = 14) Data are presented as mean values ± SD. The p-value refers to comparison of response difference in patients and controls by ANOVA for repeated measurements. *p < 0.05 in comparison with the baseline values. FMD, flow-mediated vasodilation.

	Parameter	Baseline	30 min	2 h	4 h	p-value
	Resting brachial artery diameter (mm)
	Patients	4.41 ± 0.51	4.42 ± 0.53	4.42 ± 0.55	4.41 ± 0.51	0.847
	Controls	4.80 ± 0.37	4.81 ± 0.34	4.81 ± 0.32	4.82 ± 0.36
	Resting brachial artery flow (ml min -1 )
	Patients	62 ± 31	73 ± 38*	56 ± 40	52 ± 35*	0.908
	Controls	56 ± 32	63 ± 22	52 ± 29	45 ± 27
	Hyperaemic brachial artery flow (ml min -1 )
	Patients	303 ± 113	278 ± 138	276 ± 115	294 ± 112	0.010
	Controls	286 ± 99	340 ± 106*	367 ± 124*	350 ± 124*
	FMD (%)
	Patients	7.03 ± 1.99	3.92 ± 1.78*	6.36 ± 2.12*	7.03 ± 1.99	<0.001
	Controls	8.22 ± 1.60	8.66 ± 1.88	8.64 ± 1.76	8.64 ± 1.57

Table 4

 4 Correlations between FMD values (baseline, at 30 min and 2 h after exercise) and different parameters in all individuals (n = 44)

	Baseline FMD	FMD at 30 min	FMD at 2 h

Vascular Medicine 2008; 13: 255-262

Vasodilator function and intermittent claudication 259 Vascular Medicine 2008; 13: 255-262

Acknowledgements

This study was supported by the Slovenian Research Agency, grant No. P3-0308. We would like to thank research nurses Simona Auman and Katja Janša-Trontelj and our laboratory staff for their expert assistance, and the Clinical Institute of Clinical Chemistry and Biochemistry at the University Medical Centre in Ljubljana for CRP measurements.


In this issue of Vascular Medicine, Shaffer and Mohler describe the effects of vascular disease and aging on the regulation of circulating endothelial progenitor cells. [START_REF] Schaffer | Effect of acute exercise on endothelial progenitor cells in patients with peripheral arterial disease[END_REF] This study is of interest because aging, cardiovascular risk factors, and cardiovascular diseases are all associated with reductions in endothelial function. Previous studies in healthy subjects without cardiovascular disease have shown a correlation between the number of circulating endothelial progenitor cells and the overall risk of subsequent cardiovascular events, as well as a correlation between circulating progenitor cells and endothelial function. [START_REF] Hill | Circulating endothelial progenitor cells, vascular function, and cardiovascular risk[END_REF] In patients with established cardiovascular disease, circulating progenitor cells at baseline are associated with a subsequent risk of cardiovascular events. [START_REF] Werner | Circulating endothelial progenitor cells and cardiovascular outcomes[END_REF] Thus, normal endothelial function is in part maintained by circulating endothelial progenitor cells that can interact with vascular endothelium. Impairment of endothelial function and subsequent clinical outcome is associated with decreased numbers of circulating progenitor cells. [START_REF] Berger | Aspirin for the primary prevention of cardiovascular events in women and men: a sex-specific meta-analysis of randomized controlled trials[END_REF] In addition, acute exercise is known to modulate endothelial function, [START_REF] Stewart | Exercise training for claudication[END_REF] possibly by mobilizing progenitor cells.

In the study by Shaffer and Mohler, several progenitor cell populations were measured at rest and in response to exercise in healthy control subjects and in patients with peripheral arterial disease (PAD). The results demonstrated that healthy resting younger subjects had higher progenitor cell numbers than the older or PAD groups, but the overlap in individuals was considerable. Graded exercise in young healthy subjects was associated with an increase in several progenitor cell populations, but patients with PAD and older subjects had no change or variable changes in these cell lines with exercise. The authors concluded that in addition to aging, muscle ischemia induced by exercise in patients with PAD was an insufficient stimulus to mobilize endothelial progenitor cells. Their findings may relate to loss of endothelial function in these populations.

Understanding the effects of endothelial progenitor cells on endothelial function may provide information on several potential pathways that relate to claudication therapy. For example, exercise training improves walking distance in patients with PAD, and this clinical improvement is not associated with any changes in calf blood flow. [START_REF] Hiatt | Benefit of exercise conditioning for patients with peripheral arterial disease[END_REF] Several effects of training may account for this improvement, including alterations in the biomechanics of walking, adaptations in muscle metabolism and improvements in endothelial function as assessed by the activity of nitric oxide. [START_REF] Stewart | Exercise training for claudication[END_REF]

Whether changes in circulating endothelial cells with training

Editorial

Vascular Medicine 2006; 11: 258 relate to the clinical improvement that results from exercise training will require further study.

While the findings of Shaffer and Mohler are of considerable interest, several questions remain. In particular, this study found increases in endothelial progenitor cell numbers in healthy young subjects who were tested at higher work loads than the older or PAD subjects. Therefore the exercise stimulus was not equivalent between groups. Further investigation is warranted to more critically look at the dose-response relationships between exercise duration, intensity, and mobilization of different cell lines in healthy subjects.

Despite those limitations, alterations in the number and/or function of circulating endothelial progenitor cells may have clinical ramifications for vascular health and functional capacity. Greater understanding of the role of progenitor cells in the regeneration of the endothelial lining may have therapeutic implications, particularly as it pertains to exercise training.

William R Hiatt


Gabriella V Bedarida

Ulrich Hoffmann
email: ulrich.hoffmann@med.uni-muenchen.de

Federico Tatò

Acute lower limb ischemia due to thrombo-embolic arterial occlusions in two previously healthy men with markedly elevated Lp(a)

Keywords: acute limb ischemia, arterial thrombosis, Lp(a), myointimal hyperplasia, peripheral artery disease

Acute lower limb ischemia due to thrombo-embolic arterial occlusions in two previously healthy men with

markedly elevated Lp(a)

Introduction

Elevated lipoprotein (a) (Lp(a)) is an established risk factor for atherosclerosis and has been linked to premature coronary artery disease as well as stroke and peripheral artery disease (PAD). [START_REF] Loscalzo | Lipoprotein(a). A unique risk factor for atherothrombotic disease[END_REF][START_REF] Danesh | Lipoprotein(a) and coronary heart disease: meta-analysis of prospective studies[END_REF][START_REF] Valentine | Lipoprotein (a) is an independent, discriminating risk factor for premature peripheral atherosclerosis among white men[END_REF] Lp(a) possesses unique antifibrinolytic and prothombotic properties, and may play an important role in vascular thrombosis. [START_REF] Loscalzo | Lipoprotein(a). A unique risk factor for atherothrombotic disease[END_REF][START_REF] Koschinsky | Lipoprotein (a) and the link between atherosclerosis and thrombosis[END_REF] However, elevated Lp(a) has not previously been recognized as a risk factor for acute thrombo-embolic occlusions of peripheral arteries. We report the cases of two adult men with markedly elevated Lp(a) and multifocal thrombo-embolic occlusions of the iliaco-femoral and popliteo-crural arteries in the absence of advanced atherosclerosis.

Case reports

Case 1

A 54-year-old previously healthy white male presented to our clinic with a 3-week history of right-sided calf and foot claudication. His past medical history was significant only for a recently developed impotence, for which he had received three monthly testosterone injections. He had no history of smoking, diabetes, hypertension, hypercholesterolemia, and no family history of premature cardiovascular disease.

The physical examination and non-invasive vascular laboratory tests showed bilateral, distal PAD with impaired hemodynamic function at the level of the right calf and left foot. Duplex ultrasound showed hypoechogenic occlusion of the right popliteal artery in the absence of arteriosclerosis or aneurysm. The patient underwent an extensive work-up including a 24-hour Holter monitor, transesophageal echocardiogram and aortic imaging to rule out embolic disease. Laboratory tests including markers of inflammation (ESR, CRP), electrophoresis, antinuclear and anticardiolipin antibodies and tests for thrombophilia (fibrinogen, ATIII, protein C and S, activated protein C) were normal. Homocysteine was 13.2 mol/l (normal values Ͻ13.9 mol/l). Lipid and lipoprotein analysis showed normolipidemia (total cholesterol 203 mg/dl, LDL-cholesterol 120 mg/dl, HDL-cholesterol 55 mg/dl, triglycerides 153 mg/dl). Lp(a) was measured with a commercial turbidometric immunoassay (Wako, Japan) with a normal reference range of Ͻ30 mg/dl and it was markedly elevated at 86 mg/dl.

Digital subtraction angiography (DSA) showed occlusion of the popliteal artery of the right leg (Figure 1), and occlusion of the posterior and anterior tibial arteries of the left leg with thrombo-embolic appearance. Percutaneous recanalization of the right popliteal artery was attempted by catheter-directed thrombolysis with urokinase followed by aspiration thrombectomy and percutaneous transluminal angioplasty (PTA). All procedures were unsuccessful. The patient was treated with oral anticoagulation and walking exercise. After 3 months the patient showed marked symptomatic and hemodynamic improvement. immunoassay (Wako), was markedly elevated at 81 mg/dl (normal Ͼ30 mg/dl).

DSA performed at an outside facility a few days prior to presentation showed diffuse, high-grade restenosis of the right common and external iliac artery (Figure 2B), and of the left popliteal artery. Duplex ultrasound showed circular, hypoechogenic wall thickening in the affected arteries consistent with myointimal hyperplasia. We recommended PTA of the right iliac artery. The patient wished to undergo the intervention at an outside facility and was lost to follow-up. From a telephone contact, he is experiencing persistent bilateral claudication despite repeated interventions.

Discussion

We present the cases of two men with severe PAD due to multiple, thrombo-embolic arterial occlusions in the absence of advanced arteriosclerosis and traditional cardiovascular risk factors. Markedly elevated Lp(a) above 80 mg/dl was identified as the only significant laboratory abnormality. The morphology of the occlusions and the acute onset of symptoms suggest either embolism or local arterial thrombosis. A source of embolism could not be identified. However, embolism of unknown origin remains a possibility. Other etiologies of non-arteriosclerotic PAD such as thrombangiitis obliterans, large-vessel vasculitis, or popliteal entrapment have either been ruled out or appear very unlikely based on the clinical presentation and vascular morphology.

Elevated Lp(a) is a well-established risk factor for cardiovascular disease [START_REF] Danesh | Lipoprotein(a) and coronary heart disease: meta-analysis of prospective studies[END_REF] and it has also been shown to be a strong predictor of peripheral vascular disease, independently of cigarette smoking and diabetes in a group of 100 white male patients with mean age of 67 years. [START_REF] Widmann | Lipoprotein (a): a risk factor for peripheral vascular disease[END_REF] In a case-control study of 50 white men under the age of 45 years with PAD, an elevated Lp(a) (Ͼ30 mg/dl) was an independent risk factor for the development of premature peripheral vascular disease. No association was found for homocysteine, antiphospholipid antibodies, antithrombin III, and protein C and S. [START_REF] Valentine | Lipoprotein (a), homocysteine, and hypercoagulable states in young men with premature peripheral atherosclerosis: a prospective, controlled analysis[END_REF] In a similar retrospective study in 55 white men, an elevated Lp(a) was specifically and independently associated with premature PAD but not with premature coronary artery disease. [START_REF] Valentine | Lipoprotein (a) is an independent, discriminating risk factor for premature peripheral atherosclerosis among white men[END_REF] However, patients in these studies presumably had arteriosclerotic PAD. To our knowledge this is the first report of highly elevated Lp(a) in patients with an initial diagnosis of PAD due to acute arterial thrombo-embolic occlusions.

The structural homology of apolipoprotein (a) with plasminogen confers Lp(a) unique antifibrinolytic and prothrombotic properties. [START_REF] Loscalzo | Lipoprotein(a). A unique risk factor for atherothrombotic disease[END_REF][START_REF] Koschinsky | Lipoprotein (a) and the link between atherosclerosis and thrombosis[END_REF] Several lines of evidence suggest that Lp(a) may be a risk factor for thrombotic vascular disorders unrelated to arteriosclerosis. Elevated Lp(a) has been linked to venous thrombo-embolism, 7 childhood ischemic stroke, 8 left atrial thrombus, [START_REF] Igarashi | Elevated serum lipoprotein(a) is a risk factor for left atrial thrombus in patients with chronic atrial fibrillation: a transesophageal echocardiographic study[END_REF] and pregnancy loss. [START_REF] Krause | Lipoprotein(a) and other prothrombotic risk factors in Caucasian women with unexplained recurrent miscarriage. Results of a multicentre case-control study[END_REF] Another remarkable feature of the two presented cases is the unsatisfactory outcome after percutaneous and surgical vascular interventions. Elevated Lp(a) has been previously recognized as a risk factor for shortterm, myointimal restenosis after interventions in peripheral arteries. [START_REF] Lippi | Usefulness of lipids, lipoprotein(a) and fibrinogen measurements in identifying subjects at risk of occlusive complications following vascular and endovascular surgery[END_REF] In a prospective study of 139 consecutive patients with successful PTA of the femoropopliteal area, the group who developed re-stenosis or re-occlusion within 1 year had a higher Lp(a) than the group without re-stenosis (59 vs 28 mg/dl). This was independent of all other risk factors, which were equally distributed between the two groups. [START_REF] Maca | Elevated lipoprotein (a) and increased incidence of restenosis after femoropopliteal PTA. Rationale for the higher risk of recurrence in females?[END_REF] Moreover, Lp(a) has been shown to accumulate specifically at sites of initial myointimal hyperplasia in an animal model after arterial injury caused by balloon angioplasty. [START_REF] Ryan | Localization of lipoprotein(a) in a monkey model of rapid neointimal growth[END_REF] Patient 1 received testosterone replacement therapy prior to the occurrence of claudication. We cannot exclude that the hormonal therapy was a precipitating event for the thrombotic disease. The net effect of testosterone on the cardiovascular risk profile is unknown and both beneficial and deleterious effects have been recorded. [START_REF] Wu | Androgens and coronary artery disease[END_REF] We found one report of acute limb-threatening ischemia due to thrombosis of femoropopliteal and crural arteries after treatment with the synthetic androgen danazol, which was, however, mediated by severe thrombocytosis. [START_REF] Alvarado | Danazol and limbthreatening arterial thrombosis: two case reports[END_REF] Although our findings need to be confirmed in other cases, we believe that markedly elevated Lp(a) may have played an important causative role in the presented two cases of acute PAD due to thromboembolic arterial occlusions. We suggest that it may be worthwhile to measure Lp(a) in young patients with PAD with unexplained, acute, arterial occlusions and lack of other traditional risk factors.

Case 2 A

 2 54-year-old, previously healthy white male presented at another hospital with acute, critical ischemia of the right leg. Angiography revealed occlusions of the right common and external iliac artery (Figure2A) and of the left tibio-peroneal trunk. Surgical thrombectomy of the right iliac artery required two interventions because of early re-occlusion, and was unsuccessful in the left tibio-peroneal trunk. He underwent an extensive work-up including a 24-hour Holter monitor, transesophageal echocardiogram and aortic imaging to rule out embolic disease. At the time of discharge from the hospital ankle-brachial indices were normal and walking distance unlimited. The patient was first seen at our clinic 8 months later complaining of a 5-month history of bilateral calf claudication. He had no history of diabetes, hypertension, hypercholesterolemia, and no family history of cardiovascular disease. He had quit smoking 1 year previously after a 12-pack year history. Physical examination and non-invasive vascular laboratory tests showed bilateral PAD with impaired hemodynamic function at the level of the right thigh and left calf. Laboratory tests including markers of inflammation (ESR, CRP), electrophoresis, antinuclear and anticardiolipin antibodies and tests for thrombophilia (fibrinogen, ATIII, protein C and S, activated protein C) were normal. Homocysteine was 9.2 mol/l (normal values Ͻ13.9 mol/l). Lipid and lipoprotein analysis showed a borderline hypercholesterolemia (total cholesterol 209 mg/dl, LDL-C 147 mg/dl, HDL-C 40 mg/dl, triglycerides 108 mg/dl). Lp(a), measured with a commercial turbidometric

Figure 1

 1 Figure 1 Digital subtraction angiography of the right leg in patient 1: occlusion of the popliteal artery at the level of the knee joint space and of the proximal crural vessels with thrombo-embolic appearance.

Figure 2

 2 Figure 2 Digital subtraction angiography of the aortoiliac vessels in patient 2 at the time of initial presentation (A), and 8 months after surgical revascularization (B). The first angiogram shows acute, thrombo-embolic occlusion of the right iliac arteries, and the second angiogram shows high-grade, myointimal restenosis.


Marianne Brodmann

Gerald Seinost

Ernst Pilger

HAL is

A 39-year-old white female with severe peripheral arterial disease, due to fibromuscular dysplasia, developed coumarin-induced skin necrosis.

Owing to recurrent arterial thromboses, including one episode which resulted in left above-knee amputation, phenprocoumon therapy was initiated, at doses of 15, 9 and 3 mg on the first, second and third days of treatment, respectively. This treatment overlapped with a continuous intravenous infusion of heparin that was adjusted to aPTT levels of 1.5 to 2.5 times the normal range. On the third day of treatment she had an international ratio (INR)Ͼ4.5 (normal therapeutic range 2-4.5); the protein C level was below 5%. Heparin therapy was then stopped. Twenty-four hours later she developed severe pain of the left thigh, above the site of the amputation, associated with extensive erythema. Another 24 hours later, petechial hemorrhages had developed (Panel A) and were rapidly followed by necrosis

  (Panel B). Oral anticoagulation was discontinued and © 2006 SAGE Publications 10.1177/1358863x06072229 Images in vascular medicine Coumarin skin necrosis: a severe complication of oral anticoagulation Marianne Brodmann, Gerald Seinost and Ernst Pilger Vascular Medicine 2006; 11: 273 Division of Angiology, Department of Internal Medicine, Medizinische Universität Graz, Graz, Austria Address for correspondence: Marianne Brodmann, Division of Angiology, Department of Internal Medicine, Medizinische Universität Graz, Auenbruggerplatz 15, A-8036 Graz, Austria. Tel: ϩ43 316 385 80286; Fax: ϩ43 316 385 3788; E-mail: marianne.brodmann@meduni-graz.at low molecular weight heparin was resumed. Laboratory work-up indicated normal levels of protein C, protein S, antithrombin III, lupus anticoagulant and antiphospholipid antibodies. Testing for the factor V Leiden mutation was negative, as was testing for G 20210A mutation. Within a 3-week period, the necrotic areas fully resolved spontaneously.


Teresa L Carman

Sean P Lyden

Editor in Chief, Mark A Creager

come

A 67-year-old woman with long-standing rheumatoid arthritis was admitted for bilateral femoral to anterior tibial artery bypasses for gangrenous toes. She was noted to have chronic skin pigmentation changes affecting her legs, arms and her face, which started after using minocycline (Panels A and B). Blue-black pigmentation is a well-recognized side effect of minocycline therapy. [START_REF] Eisen | Minocycline-induced pigmentation: incidence, prevention and management[END_REF] The most commonly involved sites include the skin, oral cavity, teeth, bones, and thyroid. Similar pigmentation changes were noted in the common femoral artery and anterior tibial artery walls at the time of endarterectomy and bypass surgery (Panel C). Very few reports of atherosclerotic plaque pigmentation involvement have been published. [START_REF] Gerson | Black pigmentation of atherosclerotic plaques associated with chronic minocycline therapy[END_REF][START_REF] Saint'ambrogio | Minocycline pigmentation of heart valves[END_REF] Despite the pigmented appearance of the arterial wall, uneventful revascularization was accomplished. She did eventually require amputation of her necrotic digits.

© 2006 SAGE Publications 10.1177/1358863x06073459

Images in vascular medicine

Drug-related skin and atherosclerotic plaque pigmentation

TeresaL

  Carman a and Sean P Lyden b a Cardiovascular Medicine, Section of Vascular Medicine; b Department of Vascular Surgery, Cleveland Clinic, Cleveland, OH, USA Address for correspondence: Teresa L Carman, Associate Staff, Vascular Medicine, Cleveland Clinic, 9500 Euclid Ave/S-60, Cleveland, OH 44195, USA. Tel: ϩ1 216 445 5454; Fax: ϩ1 216 444 7370; E-mail: carmant@ccf.org


Vittorio Schiano

Gregorio Brevetti
email: brevetti@unina.it

Giusy Sirico

Antonio Silvestro

Giuseppe Giugliano

Massimo Chiariello

Functional status measured by walking impairment questionnaire and cardiovascular risk prediction in peripheral arterial disease: results of the Peripheral Arteriopathy and Cardiovascular Events (PACE) study

Keywords: cardiovascular risk, functional status, intermittent claudication, peripheral arterial disease, walking impairment questionnaire

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Peripheral arterial disease (PAD) is an important manifestation of atherosclerosis affecting a relatively large proportion of the adult population. [START_REF] Fowkes | Edinburgh Artery Study: prevalence of asymptomatic and symptomatic peripheral arterial disease in the general population[END_REF][START_REF] Meijer | Peripheral arterial disease in the elderly: The Rotterdam Study[END_REF][START_REF] Hiatt | Effect of diagnostic criteria on the prevalence of peripheral arterial disease. The San Luis Valley Diabetes Study[END_REF][START_REF] Stoffers | The prevalence of asymptomatic and unrecognized peripheral arterial occlusive disease[END_REF][START_REF] Wouter | Peripheral arterial disease in the elderly. The Rotterdam Study[END_REF] Its most common symptomatic form (ie intermittent claudication) places a great burden on healthcare systems and on society as a whole. Actually, it represents an important cause of disability, [START_REF] Barletta | Quality of life in patients with intermittent claudication: relationship with laboratory exercise performance[END_REF][START_REF] Breek | Quality of life in patients with intermittent claudication using the World Health Organization (WHO) questionnaire[END_REF] and, even more importantly, is associated with an elevated risk of myocardial infarction and stroke. [START_REF] Criqui | Mortality over a period of 10 years in patients with peripheral arterial disease[END_REF][START_REF] Leng | Incidence, natural history and cardiovascular events in symptomatic and asymptomatic peripheral arterial disease in the general population[END_REF][START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF][START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF] This increased risk is widely recognized to be strongly related to the severity of PAD measured as ankle-brachial index (ABI). [START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF][START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF][START_REF] Mckenna | The ratio of ankle and arm arterial pressure as an independent predictor of mortality[END_REF] Conversely, the prognostic impact of the impaired functional status in patients with intermittent claudication is still obscure. This is particularly unfortunate, because claudicants may be severely limited in their occupational and leisure-time physical activity, [START_REF] Barletta | Quality of life in patients with intermittent claudication: relationship with laboratory exercise performance[END_REF][START_REF] Breek | Quality of life in patients with intermittent claudication using the World Health Organization (WHO) questionnaire[END_REF] and a sedentary lifestyle is a risk factor for adverse cardiovascular events. [START_REF] Leon | Leisure-time physical activity levels and risk of coronary heart disease and death. The Multiple Risk Factor Intervention Trial[END_REF][START_REF] Pate | Physical activity and public health. A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine[END_REF][START_REF] Paffenbarger | The association of changes in physical-activity level and other lifestyle characteristics with mortality among men[END_REF] In the Peripheral Arteriopathy and Cardiovascular Events (PACE) study, [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] the first survey in Italy to assess the prevalence and the natural history of symptomatic PAD in the general population, we evaluated the degree of disability of claudicant patients at baseline, using the Walking Impairment Questionnaire (WIQ), [START_REF] Regensteiner | Evaluation of walking impairment by questionnaire in patients with peripheral arterial disease[END_REF] a validated, disease-specific instrument, widely used in claudication trials. [START_REF] Hiatt | Benefit of exercise conditioning for patients with peripheral arterial disease[END_REF][START_REF] Regensteiner | Exercise training improves functional status in patients with peripheral arterial disease[END_REF][START_REF] Hiatt | Propionyl-L-carnitine improves exercise performance and functional status in patients with claudication[END_REF] The objective was to ascertain whether functional status is an independent predictor of future cardiovascular events in intermittent claudication. In this regard, we paid special attention to the confounding effect of ABI, the most powerful prognostic indicator in PAD. [START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF][START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF][START_REF] Mckenna | The ratio of ankle and arm arterial pressure as an independent predictor of mortality[END_REF] Functional status measured by walking impairment questionnaire and cardiovascular risk prediction in peripheral arterial disease: results of the Peripheral Arteriopathy and Cardiovascular Events (PACE) study Vittorio Schiano, Gregorio Brevetti, Giusy Sirico, Antonio Silvestro, Giuseppe Giugliano and Massimo Chiariello Abstract: The prognostic impact of the functional status of patients with intermittent claudication is still obscure. From the lists of seven general practitioners, we identified all subjects aged 40-80 years (n ϭ 4352). Of those reporting leg symptoms while walking on the Rose questionnaire (n ϭ 760), 60 had a qualifying diagnosis of peripheral arterial disease (PAD). All of them received the Walking Impairment Questionnaire (WIQ). For each patient affected by PAD, three sex-and age-matched controls were selected randomly. After a 24-month follow-up, survival curves showed that PAD patients with WIQ scores Ͼ median had a higher cardiovascular risk than controls, and patients with WIQ scores Ͻ median had an even poorer prognosis (p Ͻ 0.001 for all WIQ domains). In PAD, after adjustment for age, sex, ankle-brachial index and comorbidity, two WIQ domains (ie walking speed and stairclimbing) were associated with cardiovascular events. The cardiovascular risk of claudicants who had a score Ͼ median for at least three WIQ domains was intermediate versus the risk of controls and PAD patients with a WIQ score Ͻ median, also when adjusted for the covariates indicated above (RR ϭ 3.26, p ϭ 0.019). In intermittent claudication, a worse functional status entails a greater risk of ischemic events versus low functional impairment.

Methods

Participant identification

The details of the PACE study, conducted in five villages of a well-defined area of the Campania region in southern Italy, are reported elsewhere. [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] Briefly, from the lists of seven general practitioners (GPs), we identified all subjects aged 40-80 years (n ϭ 4352). GPs excluded 200 patients for the reasons reported in Figure 1. The Rose questionnaire that identifies leg symptoms that occur during walking was mailed to each subject with an explanatory letter. Of these, 287 did not answer. Of the remaining 3865 subjects, those included in the definition of 'possible' symptomatic PAD were patients reporting pain in the calf that began while walking and did not disappear while continuing to walk, and those with other complaints of the calf, foot, thigh or buttock (not of the knee) showing the ischemic pattern described above, regardless of whether the remaining Rose criteria for claudication were met (n ϭ 760). To confirm the PAD diagnosis, 'possible' symptomatic PAD cases underwent Doppler examination with assessment of the ABI and flow velocity in the femoral and posterior tibial arteries, which were measured by trained physicians as previously described. [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] Of the 760 subjects positive to the Rose questionnaire, 12 died before the vascular examination, 65 moved and 89 did not keep their appointments. Age, sex, risk factors, and prevalence of concomitant cardiovascular disease did not differ between these 166 individuals and those who underwent the vascular examination. Symptomatic PAD defined by an ABI Ͻ0.90 or reduced flow velocity in at least one leg [START_REF] Fronek | Quantitative ultrasonographic studies of lower extremity flow velocities in health and disease[END_REF] was present in 1.6% (60/3699 subjects) of the whole population. [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] The vascular examination included the administration of the WIQ to assess a patient's perceived ability to walk defined distances and speeds, ability to climb stairs, and degree of walking impairment due to severity of claudication pain. [START_REF] Regensteiner | Evaluation of walking impairment by questionnaire in patients with peripheral arterial disease[END_REF][START_REF] Hiatt | Clinical trials for claudication. Assessment of exercise performance, functional status, and clinical end points. Vascular Clinical Trialists[END_REF] Each domain has a score ranging from 0% (unable to perform the task) to 100% (no limitation). For each patient affected by PAD, three controls negative to the Rose questionnaire, and matched for sex and age (Ϯ2 years) were selected randomly from the alphabetical listing of each GP. These subjects underwent the same investigations as the PAD patients. Baseline characteristics were obtained from the subjects' electronic medical records. The software system, used by approximately 8000 Italian GPs, encodes all diagnostics records according to the ninth edition of the International Classification of Disease (ICD-9), and prescription records according to the Anatomical Therapeutical Chemical Classification system.

Hypertension was diagnosed if systolic arterial pressure exceeded 140 mmHg or diastolic arterial pressure exceeded 90 mmHg, or if the patient used antihypertensive drugs. Hyperlipidemia was diagnosed in case of plasma total cholesterol Ͼ200 mg/dl, plasma triglycerides Ͼ200 mg/dl or HDLcholesterol Ͻ35 mg/dl, or if the patient used lipidlowering drugs. Diabetes mellitus was diagnosed if plasma fasting glucose exceeded 126 mg/dl, or if the patient used hypoglycemic drugs. Smokers were defined 'former' and 'current' smokers. Furthermore, the GP completed the cumulative illness rating scale (CIRS), which explores the presence and severity of 14 diseases of major organ groups. [START_REF] Parmelee | Validation of the Cumulative Illness Rating Scale in a geriatric residential population[END_REF] Each disease group is rated as follows: 1 ϭ no, 2 ϭ mild, 3 ϭ moderate, 4 ϭ severe, 5 ϭ life-threatening. The overall illness severity was represented by the mean of the 13 CIRS items (CIRS-IS). Furthermore, a comorbidity index (CIRS-CI) was computed by counting the number of items for which moderate to severe pathology was reported (scores 3, 4, or 5). All participants gave their written informed consent to the study, which was approved by our institutional ethics committee.

Prospective follow-up

GPs were contacted to determine the occurrence of events at 6-month intervals. The occurrence of cardiac (fatal and non-fatal myocardial infarction, unstable angina, and coronary revascularization procedures), cerebrovascular (fatal and non-fatal stoke, transitory ischemic attack, and carotid surgery), and peripheral (critical limb ischemia and revascularization procedures of the lower limbs) events were prospectively assessed. Peripheral revascularization was defined as the need for surgical or percutaneous transluminal intervention for symptomatic deterioration occurring at least 1 year after the study entry. Information about these events was extracted from the patients' electronic medical records which are updated by the GP on the basis of hospital records and death certificates. Two cardiologists at our department performed a blinded review of the events to confirm the agreement between the diagnosis obtained from the hospital record and death certificate and the ICD code. For patients experiencing more than one event, only the first was considered in the analysis.

Statistical analysis

To evaluate the impact of functional status on cardiovascular risk in PAD, each domain of the WIQ was divided into scores below and above the median. The incidence of cumulative cardiovascular events in the two groups divided according to WIQ score was estimated by survival curves, and probability values were calculated by the Wilcoxon test. Cox proportional hazard regression models were then used to control for confounders identified by univariate analysis (inclusion criteria p Ͻ 0.1). [START_REF] Cox | Regression and life tables[END_REF] Survival curves were also constructed for PAD patients with a score Ͻ the median for at least three of the four WIQ domains, and in those with less impaired global functional status. Cox proportional hazard regression models were then used to control for age, sex, ABI and CIRS-IS (ie confounders identified at the univariate analysis). Values are expressed as median and interquartile range. Baseline characteristics were compared with the Mann-Whitney U test, or the 2 test, as appropriate.

Results

Figure 1 shows the flow diagram of the study and the reasons why some subjects were excluded. The baseline characteristics of the study population are detailed elsewhere. [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] Briefly, compared with controls, the PAD group included a significantly higher number of smokers (44% vs 65%, p ϭ 0.029), hypertensives (61% vs 80%, p ϭ 0.007) and diabetics (22% vs 47%, p ϭ 0.001). No group difference was observed for age, sex distribution, hyperlipidemia and both CIRS-CI (median [interquartile range]) (2.0 [1.0; 3.0] in controls, 2.0 [1.0; 4.0] in PAD patients; p ϭ 0.123) and CIRS-IS (1.6 [1.4; 1.9] in controls, 1.7 [1.4; 2.0] in PAD patients; p ϭ 0.307). Follow-up data were obtained for all intermittent claudication patients. Conversely, nine controls could not be contacted after the baseline examination. Compared with the other controls, these individuals did not differ appreciably for age, sex, risk factors and cardiovascular comorbidity. As Table 1 shows, after a median period of 24 months, a cardiovascular event was experienced by 18 (10.8%) controls (in three it was fatal) and 20 (33.3%) PAD patients (in five it was fatal). After adjustment for smoking, hypertension, diabetes mellitus, previous myocardial infarction, and previous stroke (confounders identified by univariate analysis), independent predictors of a cardiovascular event were symptomatic PAD (relative risk (RR) 2.65; 95% confidence interval (CI) 1.24-5.69; p ϭ 0.012), and previous myocardial infarction (RR 4.00; 95% CI 1.34-11.91; p ϭ 0.013). Figure 2 shows event-free survival curves for control subjects, PAD patients with a score Ͼ median for each of the four WIQ domains, and patients with a more compromised functional status (WIQ scores Ͻ median). In PAD patients, WIQ scores Ͼ median were associated with a higher incidence of cardiovascular events compared with controls, and WIQ scores Ͻ median with an even poorer prognosis. Group differences were significant for all the four WIQ domains.

In the PAD patient group, no significant differences in age, sex distribution and prevalence of both classic risk factors and previous cardiovascular events were observed between those with and without events at follow-up (Table 2). Similarly no group differences were observed with respect to the use of antiplatelets, statins, ACE inhibitors and other cardiovascular drugs. Conversely, a significant difference was observed for ABI, which was 0.44 (0.60; 0.67) in patients with, and 0.70 (0.62; 0.78) (p ϭ 0.007) in those without events. As reported in Table 2, group difference in CIRS-IS approached the statistical significance, while CIRS-CI was similar in the two groups. In the 60 PAD patients, after adjustment for age and sex, the ABI and three WIQ domains (ie walking distance, walking speed and stair-climbing) were significantly associated with cardiovascular events (Table 3). Of note, both walking speed score and stair-climbing score Ͻ median, maintained their predictive value also after adjustment for ABI and CIRS-IS (ie the variables showing significant differences at the univariate analysis) (Table 3). Figure 3 shows event-free survival curves for control subjects and PAD patients categorized as being above or below the median value for at least three WIQ domains. The cardiovascular risk of PAD patients who had a score Ͼ median for at least three WIQ domains was intermediate versus the response of controls and patients with a more impaired functional status (ie WIQ score Ͻ median) (p Ͻ 0.001), also when peripheral events were excluded (p Ͻ 0.001).

Cox analysis showed that a more impaired functional status remained significantly associated with an increased cardiovascular risk, also when adjusted for age, sex, ABI and CIRS-IS (RR ϭ 3.26, 95% CI 1.22-4.74; p ϭ 0.019).

Discussion

As a result of a large number of observational epidemiologic studies, physical inactivity and a sedentary lifestyle have been recognized as risk factors for the development or progression of coronary artery disease (CAD) and for adverse cardiovascular events and death. [START_REF] Leon | Leisure-time physical activity levels and risk of coronary heart disease and death. The Multiple Risk Factor Intervention Trial[END_REF][START_REF] Pate | Physical activity and public health. A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine[END_REF][START_REF] Paffenbarger | The association of changes in physical-activity level and other lifestyle characteristics with mortality among men[END_REF] The results of the present study confirm the poor overall prognosis for individuals with symptomatic PAD, and first demonstrate an increasingly poor prognosis with increasing impairment in functional status. Actually, PAD patients with a reduced walking speed, a greater limitation in stair-climbing, and a score below the median for at least three WIQ domains showed a nearly threefold increased risk of developing a cardiovascular event, as compared with those with a less compromised functional status. Of note, the predictive value of WIQ was independent of the ABI, the most powerful prognostic indicator in intermittent claudication. [START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF][START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF][START_REF] Mckenna | The ratio of ankle and arm arterial pressure as an independent predictor of mortality[END_REF] Furthermore, the results appeared to be not affected by other factors that may influence the cardiovascular risk. Actually, the incidence of cardiovascular events was independent from traditional risk factors, history of previous myocardial infarction or stroke (the prevalence of these factors was similar in PAD patients with and without events at the follow-up), and from the CIRS-IS, a comorbidity index which explores the presence and severity of 14 diseases of major organ groups. [START_REF] Parmelee | Validation of the Cumulative Illness Rating Scale in a geriatric residential population[END_REF] This is in line with previous studies indicating that, in claudicants, cardiovascular risk appears to be independent of risk factors, and is poorly related to the expected association of PAD with coronary and cerebrovascular disease. [START_REF] Criqui | Mortality over a period of 10 years in patients with peripheral arterial disease[END_REF][START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF] Moreover, no group differences were observed with respect to the use of antiplatelet therapy and interventions aimed at correcting risk factors.

Previous data of the PACE study [START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] showed that the prevalence of PAD in Italy tended to be lower than that in northern American and northern European countries. [START_REF] Fowkes | Edinburgh Artery Study: prevalence of asymptomatic and symptomatic peripheral arterial disease in the general population[END_REF][START_REF] Meijer | Peripheral arterial disease in the elderly: The Rotterdam Study[END_REF][START_REF] Hiatt | Effect of diagnostic criteria on the prevalence of peripheral arterial disease. The San Luis Valley Diabetes Study[END_REF][START_REF] Stoffers | The prevalence of asymptomatic and unrecognized peripheral arterial occlusive disease[END_REF][START_REF] Wouter | Peripheral arterial disease in the elderly. The Rotterdam Study[END_REF] This might be due to a less aggressive nature of the atherogenic factors and/or to a lower susceptibility to them in the Italian population. However, we found that the cardiovascular risk of our claudicants was similar to that in Anglo-Saxon [START_REF] Criqui | Mortality over a period of 10 years in patients with peripheral arterial disease[END_REF][START_REF] Leng | Incidence, natural history and cardiovascular events in symptomatic and asymptomatic peripheral arterial disease in the general population[END_REF][START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF][START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF] populations. It is difficult to explain the discrepancy we observed in our population between the low symptomatic PAD prevalence and the elevated cardiovascular risk of the affected individuals. However, it may be hypothesized that, in claudicants, the various atherogenic factors are important in the development of atherosclerotic disease in the lower limbs, but scarcely influence the progression of the atherosclerosis in other vascular districts. In this regard, it is interesting to note that smoking and diabetes mellitus, which are important risk factors for the development of symptomatic PAD, [START_REF] Newman | Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group[END_REF][START_REF] Murabito | Intermittent claudication. A risk profile from The Framingham Heart Study[END_REF] scarcely influence the systemic cardiovascular risk in claudicants, as observed in this and previous studies. [START_REF] Criqui | Mortality over a period of 10 years in patients with peripheral arterial disease[END_REF][START_REF] O'riordain | Realistic expectations for the patient with intermittent claudication[END_REF] A limitation of the study is that we cannot be sure that all non-fatal events were recorded by the GPs, but given the changes in therapy and requests for laboratory examinations contained in the electronic records, it is likely that non-notified events were negligible. One of the strengths of our study is that all Italians are registered with a GP and so the population of a general practice is a segment of the general population. Other features of our study are the objective determination of PAD, follow-up examinations every 6 months, and follow-up information available for all the claudicant patients.

Beyond its novelty (to the best of our knowledge, there are no previous reports on the relationship between functional status and cardiovascular risk in PAD), our study may have important implications. First, it indicates that WIQ, which requires only few minutes to administer, could be included among the measures used to gauge cardiovascular risk in claudicants. Second, because ABI determination is more time-consuming than the self-administered questionnaire, and requires training of the observers, the WIQ could prove to be very useful in epidemiologic studies involving GPs. In this context, it should be noted that WIQ is a subjective measure of functioning. However, the repeatability and validity of the questionnaire has been documented. [START_REF] Regensteiner | Evaluation of walking impairment by questionnaire in patients with peripheral arterial disease[END_REF] Exercise training improves prognosis in diabetics 26 and patients with coronary artery disease, [START_REF] Taylor | Exercise-based rehabilitation for patients with coronary heart disease: systematic review and meta-analysis of randomized controlled trials[END_REF] who are highly prevalent in the PAD population. [START_REF] Fowkes | Edinburgh Artery Study: prevalence of asymptomatic and symptomatic peripheral arterial disease in the general population[END_REF][START_REF] Meijer | Peripheral arterial disease in the elderly: The Rotterdam Study[END_REF][START_REF] Brevetti | Peripheral Arteriopathy and Cardiovascular Events (PACE) Study Group. Prevalence, risk factors and cardiovascular comorbidity of symptomatic peripheral arterial disease in Italy[END_REF] However, there are no data about the effect of exercise on cardiovascular risk in intermittent claudication. This is particularly unfortunate because in these patients exercise increases the capacity to perform daily physical activities, [START_REF] Hiatt | Benefit of exercise conditioning for patients with peripheral arterial disease[END_REF][START_REF] Regensteiner | Exercise training improves functional status in patients with peripheral arterial disease[END_REF] favorably modifies the cardiovascular risk factors profile, [START_REF] Izquierdo-Porrera | Effects of exercise rehabilitation on cardiovascular risk factors in older patients with peripheral arterial occlusive disease[END_REF] and improves endothelial function. [START_REF] Brendle | Effects of exercise rehabilitation on endothelial reactivity in older patients with peripheral arterial disease[END_REF] Thus, there is a need for studies aimed at verifying whether exercise training may reduce cardiovascular risk in patients with symptomatic PAD.

Figure 1

 1 Figure 1 Flow diagram of the PACE study. Reasons for non-participation and exclusion.

Figure 2

 2 Figure 2 Cardiovascular event (fatal plus non-fatal)-free survival curves refer to control subjects and to PAD patients with scores above and below the median for each of the four walking impairment questionnaire (WIQ) domains.

  WIQ and cardiovascular risk prediction in PAD 151Vascular Medicine 2006; 11: 147-154

Figure 3

 3 Figure 3Cardiovascular event (fatal plus non-fatal)-free survival curves refer to control subjects and to PAD patients who had a score Ͼ the median for at least three walking impairment questionnaire (WIQ) domains and patients with greater functional impairment. Left panel: all cardiovascular events; right panel: all events except peripheral events.

Table 1

 1 Cardiovascular mortality and cardiac, cerebrovascular and peripheral events observed during follow-up.

		PAD	Controls	Adjusted RR
		(n ϭ 60)	(n ϭ 167)	(95% CI)	p
	Cardiovascular mortality	5 (8.3%)	3 (1.8%)	7.77 (1.51-40.16)	0.014
	Cardiac events	11 (18.3%)	6 (3.6%)	4.33 (1.46-12.82)	0.008
	Sudden death	2 (3.3%)	1 (0.6%)
	Myocardial infarction	2 (3.3%)	1 (0.6%)
	Fatal	1 (1.6%)	0 (0%)
	Coronary revascularization	2 (3.3%)	1 (0.6%)
	Angina	5 (8.3%)	3 (1.8%)
	Cerebrovascular events	5 (8.3%)	11 (6.6%)	1.13 (0.32-4.01)	0.844
	Stroke	3 (5.0%)	3 (4.8%)
	Fatal	2 (3.3%)	2 (1.2%)
	TIA	2 (3.3%)	8 (4.8%)
	Carotid revascularization	0 (0%)	0 (0%)
	Peripheral events	4 (6.6%)	1 (0.6%)	9.51 (1.01-91.49)	0.043
	Peripheral revascularization	4 (6.6%)	0 (0%)
	Critical limb ischemia	0 (0%)	1 (0.6%)
	Amputation	0 (0%)	0 (0%)

PAD, peripheral arterial disease; RR, relative risk; CI, confidence interval; TIA, transient ischemic attack.

Table 2

 2 Baseline characteristics of symptomatic PAD patients with and without a cardiovascular event at follow-up.

		Cardiovascular events
		With (n ϭ 20)	Without (n ϭ 40)	p
	Clinical characteristics
	Age (years)	67.0 (61.0; 76.0)	70.0 (63.5; 77.5)	0.410
	Men, n (%)	15 (75)	26 (65)	0.432
	Smokers, n (%)	12 (60)	25 (63)	0.961
	Hypertension, n (%)	15 (75)	32 (80)	0.658
	Diabetes mellitus, n (%)	10 (50)	20 (50)	1.000
	Hyperlipidemia, n (%)	11 (55)	27 (68)	0.220
	Previous MI, n (%)	4 (20)	3 (8)	0.155
	Previous stroke, n (%)	2 (10)	1 (3)	0.218
	CIRS-CI	3.0 (1.0; 5.0)	2.0 (1.0; 4.0)	0.375
	CIRS-IS	1.9 (1.5; 2.2)	1.7 (1.4; 1.5)	0.082
	ABI	0.44 (0.60; 067)	0.70 (0.62; 0.78)	0.007
	Treatments
	␤-blockers (%)	0 (0)	2 (5)	0.223
	ACE inhibitors (%)	11 (55)	16 (40)	0.271
	Antiplatelets (%)	13 (65)	22 (55)	0.459
	Lipid-lowering drugs (%)	5 (25)	7 (18)	0.494
	WIQ domains
	Claudication pain	50.0 (25.0; 75.0)	75.0 (50.0; 75.0)	0.370
	Walking distance	9.0 (2.0; 46.0)	34.0 (12.0; 75.0)	0.022
	Walking speed	6.0 (3.0; 20.0)	22.0 (7.0; 36.0)	0.046
	Stair climbing	12.0 (0.0; 42.0)	54.0 (19.0; 75.0)	0.012

MI, myocardial infarction; CIRS-CI, cumulative illness rating scale -comorbidity index; CIRS-IS, cumulative illness rating scale -illness severity; ABI, ankle-brachial index; ACE, angiotensin converting enzyme; WIQ, walking impairment questionnaire. Data presented as median (interquartile range).

Table 3

 3 Predictive value of ABI and WIQ domains in PAD patients.

			Cox proportional hazard models
					Adjusted for
		Adjusted for age and sex		age, sex, ABI and CIRS-IS
		RR	95% CI	p	RR	95% CI	p
	ABI Ͻ Ͻ median	3.57	1.26-10.13	0.017
	WIQ domains
	Claudication pain Ͻ Ͻ median	1.10	0.43-2.86	0.840	0.92	0.36-2.35	0.861
	Walking distance Ͻ Ͻ median	2.84	1.07-7.49	0.035	2.30	0.84-6.33	0.105
	Walking speed Ͻ Ͻ median	3.92	1.46-10.03	0.007	3.73	1.33-10.42	0.012
	Stair-climbing Ͻ Ͻ median	3.61	1.30-10.03	0.014	2.94	1.04-8.35	0.043
	ABI, ankle-brachial index; WIQ, walking impairment questionnaire; PAD, peripheral arterial disease; CIRS-IS,
	cumulative illness rating scale -illness severity; RR, relative risk; CI, confidence intervals.


come

Popliteal venous entrapment syndrome

A 27-year-old previously healthy male presented with a 2-month history of persistent, unexplained pain and swelling within his right calf. His signs and symptoms worsened with exercise and only partially improved with elevation. An examination was remarkable for a swollen, mildly tender right calf measuring 3 cm larger in diameter than the left calf. An extensive evaluation prior to his assessment was unrevealing and included three venous duplex ultrasounds, a triple phase bone scan, plain film radiography, two MRIs (one with magnetic resonance venography), and lab work ( Although less recognized than popliteal artery compression, popliteal vein compression has been documented in up to 27% of healthy subjects. [START_REF] Leon | Popliteal vein entrapment in the normal population[END_REF] In one study, popliteal vein constriction with active or passive ankle flexion was incidentally identified in 42% of patients who were subjected to ascending venography for various reasons. [START_REF] Raju | Popliteal vein entrapment: a benign venographic feature or a pathologic entity[END_REF] Popliteal venous compression is usually a benign and physiological finding; however, pathological popliteal 'Images in vascular medicine' is a regular feature of Vascular Medicine. Readers may submit original, unpublished images related to clinical vascular medicine to: Mark A Creager, Editor in Chief, Vascular Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.

vein entrapment syndrome should be considered when typical causes of infrageniculate pain and swelling have been excluded. Limited data indicate that popliteal vein release surgery is often palliative or even curative.

  University, Division of Cardiovascular Medicine, 200 Dorothy Davis Heart and Lung Research Institute, Columbus, Ohio, USA Address for correspondence: Steven M Dean, Vascular Medicine, Assistant Professor of Clinical Internal Medicine, The Ohio State University, Division of Cardiovascular Medicine, 200 Dorothy Davis Heart and Lung Research Institute, 473 W 12th Avenue, Columbus, OH 43210, USA. Tel: +1 614 293 4967; Fax: +1 614 293 5614; E-mail: steven.dean@osumc.edu Panel A Panel B D-dimer). Consequently, a right lower extremity ascending venogram in neutral and stress positions was ordered. A venographically normal right popliteal vein was observed with the limb in a neutral position (Panel A). Venography performed during active plantar flexion of the foot provoked elongated popliteal vein occlusion (Panel B). The patient ultimately underwent popliteal vein entrapment release; subsequently, his pain resolved and his swelling markedly improved.


Frank Schröder

Ingrid Hausser

Christiane Szliska

Holger Lawall

Curt Diehm

Pseudoxanthoma elasticum: under-recognized cause of early onset peripheral arterial disease?

A 50-year-old female patient with critical limb ischemia was referred for evaluation and treatment of peripheral arterial disease (PAD). She had a history of stroke in 2002; however, no classical cardiovascular risk factor was detected. Angiography revealed a severe generalized PAD (Panel A). Small, yellowish, flat, soft papules were present in the neck (Panels B and C) and both antecubital fossae. A skin biopsy showed typical calcified elastic fibres (Panel D: part C) and disintegrated and fringed collagen fibrils (Panel D: parts A and B). These findings are consistent with pseudoxanthoma elasticum (synonym: PXE, Grönblad-Strandberg syndrome). It is a systemic inherited disease with a prevalence of 1:25 000-1:100 000 in white individuals. [START_REF] Terry | Pseudoxanthoma elasticum[END_REF] The defect has been mapped to the ABCC6 gene on chromosome 16 encoding multidrug resistance-associated protein. [START_REF] Saux | Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum[END_REF] It causes fragmentation and calcium deposition in elastic fibres of skin, blood vessels, Bruch's membrane of the eye, and other organs. It is not known how this mutation causes these changes in multiple organs. One hypothesis is chronic oxidative stress. [START_REF] Pasquali-Ronchetti | Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations[END_REF] Characteristically, patients suffer from skin lesions, cardiovascular manifestations such as early PAD, myocardial infarction or stroke, and ophthalmologic features (i.e. angioid streaks, peau d'orange and maculopathy). The anticipated prevalence in Germany is at least 1000 patients. However, only a few hundred are diagnosed. Therefore, the disease seems to be under-recognized. Pseudoxanthoma elasticum should be considered as a possible diagnosis in patients with early onset of PAD.

Images in vascular medicine

  Pseudoxanthoma elasticum: under-recognized cause of early onset peripheral arterial disease? Frank Schröder a , Ingrid Hausser b , Christiane Szliska c , Holger Lawall a and Curt Diehm a a Klinikum Karlsbad-Langensteinbach, Karlsbad, Germany; b Universitäts-Hautklinik der Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany; c Krankenaus Bethesda, Dermatologische Klinik, Freudenberg, Germany Address for correspondence: Frank Schröder, Klinikum Karlsbad-Langensteinbach, Teaching Hospital of the Ruprecht-Karls-University Heidelberg, D-76307 Karlsbad, Germany. E-mail: Schroederhester@yahoo.de Panel A