Assessment of endothelial damage in atherosclerotic vascular disease by quantification of circulating endothelial cells

Relationship with von Willebrand factor and tissue factor

Andrew J Makina,b, Andrew D Blanna, Natali A.Y Chunga, Stanley H Silvermanb and Gregory Y.H Lipa,*

a Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, UK
b Department of Vascular Surgery, City Hospital, Birmingham B18 7QH, UK

* Corresponding author. Tel.: +44-121-507-5080; fax: +44-121-554-4083
E-mail address: g.y.h.lip{at}bham.ac.uk

Received 21 October 2002; accepted 7 April 2003

Abstract

Background Increased numbers of CD146-defined circulating endothelial cells (CECs), as are present in the peripheral blood of patients suffering acute coronary syndromes, imply injury to the endothelium. Endothelial damage can also be assessed by the measurement of plasma levels of von Willebrand factor (vWf). Increased levels of procoagulant plasma tissue factor (TF), arising from monocytes/macrophages and endothelial cells, is present in atherosclerosis. We hypothesised increased CECs in patients with ischaemic rest pain (IRP) of the lower limb due to peripheral atherosclerosis and comparable to that seen in patients with acute myocardial infarction (AMI), when compared to patients with intermittent claudication (IC) or healthy controls that would correlate with vWf and TF.

Patients and methods We recruited 20 patients in each of four groups: (i) IRP of the lower limb; (ii) AMI; (iii) ‘stable’ IC; and (iv) healthy controls. CD146-expressing CECs were measured by immumomagnetic separation and counting under a fluorescence microscope; plasma vWf and TF by ELISA.

Results In IRP, median (IQR) CEC levels were 3.5 (2.0–5.8) cells/ml, in IC were 1.1 (0.6–2.9) cells/ml, and in healthy controls were 1.0 (0.5–1.7) cells/ml (). The levels of vWf () and TF () were also significantly different between the groups, with the highest levels in patients with IRP. Levels of CECs correlated with vWf (, ) and TF (, ). In AMI, CEC levels were higher than those in IRP at 4.9 (3.6–8.4) cells/ml ().

Conclusion This study demonstrates evidence of direct endothelial cell injury (i.e. raised CECs) in patients with IRP that correlated with vWf and TF, but that this is less severe than in AMI.

Key Words: Circulating endothelial cells • von Willebrand factor • Ischaemic rest pain • Peripheral atherosclerosis • Intermittent claudication • Tissue factor

Introduction

The loss of adequate vascular function and endothelial damage are important in the pathogenesis of atherosclerosis and thus peripheral artery disease (PAD).1,2 The latter is associated with considerable morbidity and mortality, especially when the condition progresses to the so-called ‘critical ischaemia’ or ischaemic rest pain (IRP) that is characterised by the onset of gangrene or the patient complaining of pain at rest (Fontaine Stages III and IV).3 The earlier symptomatic form of this disease, intermittent claudication (Fontaine Stage II), is also associated with an increase in cardiovascular mortality. Disruption of the endothelium and exposure of the blood constituents to a thrombogenic surface may in part account for the triad of the prothrombotic or hypercoagulable state seen in atherosclerosis, i.e., abnormally turbulent blood flow, pro-thrombotic blood constituents (e.g. platelets and clotting factors), and abnormalities in endothelial physiology.4

Endothelial damage can be assessed in several ways. Measurement of plasma von Willebrand factor levels has long been regarded as an index of endothelial damage/dysfunction,5 and raised von Willebrand factor has been demonstrated in both coronary and peripheral atherosclerotic disease6 and predicts an adverse outcome,7 although the relationship to disease severity is subject to debate.1,8 A further index of the intravascular prothrombotic state is tissue factor (TF) as under normal physiological conditions, this procoagulant is expressed only on extravascular sites and perivascularly in the adventitial layer of blood vessels and potentially some activated monocytes.9 However, raised plasma TF levels, possibly arising from the endothelium and/or monocytes, have been reported in patients with proven PAD and ischaemic heart disease when compared to controls.10,11 TF may well be directly implicated in the processes leading to thrombosis as, if the endothelial lining of the blood vessel is damaged and shed, this would lead to exposure of the underlying matrix and the presence of increased tissue factor in the circulating blood.

A further, recently developed, index of endothelial damage is the quantification of immunologically-defined circulating endothelial cells (CECs). Using this technique, we have demonstrated abnormal levels of CECs in patients with acute coronary syndromes and acute myocardial infarction (AMI), whereas those with ‘stable’ effort angina and healthy controls had no significant increase in peripheral blood CECs.12 Importantly, these CECs are predominantly of macrovascular origin and are not apoptotic, as suggested by intact DNA and cytoplasmic properties.12 In this setting, CECs are defined using an endothelial specific antigen CD146, a molecule associated with cell–cell junctions and the actin cytoskeleton.13 However, more recent work has raised the possibility that the CECs we define may also include a population of (or, in fact, be) haematopoietic progenitor cells that also contain endothelial precursor cells (i.e. angioblasts).14,15

In the present report, we hypothesised that levels of CECs would be greater in patients with ischaemic rest pain (IRP) of the lower limb due to peripheral atherosclerotic vascular disease, when compared to patients with intermittent claudication (IC) or healthy controls. Additionally, we hypothesised that numbers of CECs would correlate with plasma von Willebrand factor, marking endothelial cell damage, and with plasma tissue factor, an initiation of coagulation, and that raised CECs in IRP would be comparable with those from patients who recently suffered an acute myocardial infarction (AMI). We also sought to make additional phenotype findings of these cells, with respect to vascular precursor status. These hypotheses were tested by measuring CECs, von Willebrand factor and tissue factor in blood from patients with PAD (IRP or IC) or AMI, and in age and sex-matched controls.

Materials and methods

Subjects
Patients with intermittent claudication (IC) and IRP (Fontaine classes II and II/IV, respectively)3 were recruited from the vascular surgical inpatient ward and vascular surgical outpatients at City Hospital, Birmingham, UK. The diagnosis of PAD was confirmed in all patients with an ankle brachial pressure index (ABPI) 0.8. All patients were recruited prior to any angiographic intervention. We recruited patients with AMI within 24 hours of admission to the hospital Coronary Care Unit. AMI was diagnosed by the presence of the following three criteria: (i) history of typical chest pain; (ii) diagnostic ECG changes of AMI (ST elevation 2 mm in two concomitant leads); and (iii) CK level greater than twice the upper limit of normal (150 IU/dl in our laboratory) or CKMB level 10% of the total CK.

The healthy control subjects were recruited from healthy hospital staff, relatives of the patients and those attending the hospital for routine procedures such as inguinal hernia repair and cataract surgery. The subjects had no clinical evidence of vascular, metabolic, neoplastic, or inflammatory disease, by careful history, examination and routine laboratory tests. These subjects were normotensive and in sinus rhythm, and were not taking aspirin, warfarin, lipid-lowering or anti-hypertensive drugs, NSAIDs or antibiotics. Clinical and demographic details of the subjects are presented in Table 1. Informed consent was obtained from all participants, and the study was approved by the Local Research Ethics Committee.


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Table 1 Baseline demography in patients and healthy controls

 
Blood samples and laboratory methods
Blood collection was performed by atraumatic puncture of the antecubital vein. 4 ml of blood was collected into EDTA/NaF tube for CEC quantification (see below) and further blood was collected onto citrate tubes, centrifuged at 3000 rpm/1000g for 20 min and frozen at –70 °C for batch analysis of von Willebrand factor and tissue factor levels using commercial ELISAs (for von Willebrand factor, Dako, Copenhagen, Denmark; for tissue factor, Axis-Shield, Dundee, UK). The intra-assay coefficient of the ELISAs is 5% and inter-assay coefficient, 10%. All laboratory work was performed in blinded fashion with respect to the identity of the samples.

Bead preparation and immunomagnetic separation of CECs
Monodispersed magnetised 4.5 µm diameter polystyrene beads with anti-mouse IgG covalently bound to the surface (Dynabeads M-450, Dynal A.S., Oslo, Norway)10 were coated with a secondary layer of S-Endo 1, a monoclonal antibody raised against human umbilical vein endothelial cells (HUVECs) (Biocytex, Marseille, France). To coat the beads with the antibody the following process was adopted: 125 µl (approximately beads) of the raw beads were diluted with 1 ml of phosphate-buffered saline (PBS) solution and then placed in a small magnet (MPC-S, Dynal). The supernatant was gently pipetted off. This washing process was then repeated a further three times. Following the last washing, 100 µl of the S-Endo 1 antibody solution was added to the bead suspension, which was incubated and gently agitated at room temperature for one hour. This was then washed (as described above) four times. Following the last wash 1000 µl of PBS was added and the beads re-suspended and stored for a maximum of 14 days.

For measurement of CECs, 4 ml of patients or controls venous blood was diluted with a further 4 ml of phosphate-buffered saline (PBS). 100 µl of the suspension of beads coated with S-Endo 1 ( beads) were then added to the diluted blood. This mixture was incubated at room temperature for 30 min whilst being gently rotated (30 rpm) to ensure continued mixing. The rosetted beads were separated from the blood using an MPC-L concentrator (Dynal, as above) and washed a total of four times. The resulting rosetted cells and beads were finally re-suspended in 30 µl of PBS for counting by a single observer (AM) under epifluorescence microscopy (Zeiss). CECs are easily located as they are autofluorescent. The criteria for confirmation of a CEC was >=4 rosetted beads and a cell size of greater than 20 µm diameter (approximately four times bead diameter). For aggregated cells, the aggregate was counted as a single cell. Intra- and inter-assay () coefficients of variation were 5% and 10%, respectively. The inter- and intra-observer variations of the method in our laboratory were 5%.

Quality control and identity of CECs
Quality control of this method was confirmed using HUVECs obtained using standard tissue culture techniques.16 A known number of primary un-passaged HUVECs derived from a healthy confluent monolayer were removed by EDTA/trypsin and seeded into 4 ml of whole blood from a healthy individual and CECs quantified as described above. With an innoculum of 100 cells into 4 ml of blood it was possible to capture and detect 95% of cells. Further identity of CECs was performed by immunocytochemistry. Autofluorescence of air-dried glass slide preparations of CECs was quenched by 2 min incubation with Gill 3 haematoxylin (Surgipath Europe, Peterborough, UK) with no post-staining differentiation. Slides were then incubated with mouse anti-human CD34 or mouse anti-human CD31 (PECAM-1), both conjugated to fluorescein isothiocyanate (Serotec, Kidlington, UK), diluted 1/10 in PBS for 30 min. Excess antibodies were removed with three washes with PBS, and cells examined as described above.

Power calculations
We have previously reported increased CECs in the plasma of 26 subjects with AMI and 33 with unstable angina compared to 13 with stable angina and 14 healthy controls with an overall F statistic of 16 giving .12 Consequently, we hypothesised similar levels and distribution in IRP (perhaps analogous to AMI) compared to IC (perhaps analogous to stable angina) versus controls. Thus, with three groups our power calculation required 20 subjects per group (median values 8, 0 and 0) to generate a similar F statistic at . This number of subjects () provides the power to detect a correlation coefficient of 0.35 at and .17

Statistical analysis
Categorical data were compared using the test. Continuous data were subjected to the Ryan–Joiner test to assess distribution. Age and von Willebrand factor levels were normally distributed and are expressed as mean and standard deviation (SD). CEC and tissue factor levels are not normally distributed and are therefore shown as median and inter-quartile range (IQR) but raw data was logarithmically transformed for analysis. Correlations between the measured parameters were assessed according to Spearman’s method. Differences between three groups (IRP, IC, controls) were compared by one-way ANOVA, and inter-group comparisons using Tukey’s post hoc test. Data between IRP and AMI was compared using the Mann–Whitney U test or t test. All statistical calculations were performed on a microcomputer using a commercially available statistical package (SPSS 10.0 for Windows, Chicago, IL, USA). A value 0.05 was considered statistically significant.

Results

Clinical and demographic details
Baseline characteristics of all participants are given in Table 1. Patients with AMI were slightly younger than the other patient groups (one-way ANOVA, ) but the gender and risk factors were not significantly different. Healthy controls aside, there were no significant differences in the proportions of treated hypertensives, hyperlipidaemics (total cholesterol 6.5 mmol/L), smokers and diabetics in the patient groups ( test, , 0.43, and 0.735, respectively).

Peripheral artery disease and healthy controls
In IRP, median (IQR) CEC levels were 3.5 (2.0–5.8) cells/ml, in IC they were 1.1 (0.6–2.9) cells/ml, and in the healthy controls, levels were 1.0 (0.5–1.7) cells/ml (ANOVA, , ). CECs were higher in IRP than both IC and the healthy controls (, Tukey’s post-hoc test) (Fig. 1). Mean [SD] von Willebrand factor was 134 [25] IU/dL in IRP, 120 [25] IU/dL in IC and was 112 [26] IU/dL in the healthy controls (). Levels in IRP were higher than in the controls (). Median/IQR levels of tissue factor were 42 (25–95) ng/mL in IRP, 23.5 (11–50) ng/mL in IC and were 19 (16–31) ng/mL in the controls (). Levels were higher in IRP compared to the controls (). The Spearman correlation between CECs and tissue factor was moderately significant (, ), but there was a highly significant correlation between levels of CECs and von Willebrand factor (, ) (Fig. 2). Levels of tissue factor correlated with those of von Willebrand factor (, ).



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Fig. 1 Circulating endothelial cells in patients with peripheral vascular disease and in healthy controls. IC, intermittent claudication, and IRP, ischaemic rest pain. The bar represents the median value.

 


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Fig. 2 Correlation between CECs and von Willebrand factor. (, ).

 
Quality control and identify of CECs
Photomicrograph Fig. 3(a) shows the results of purifying the HUVECs that were seeded into whole blood. Note the large number of beads (median perhaps 7.5 beads/cell) rosetted around each HUVEC. Fig. 3(b) shows one CEC rosetted with 5 beads and two unrosetted cells (that seem likely to be leukocytes) purified from the whole blood of a patient with IRP. Fig. 3(c) shows a single CEC with at least 9 rosetted beads, prepared from the whole blood of a patient with IRP. Identify of CECs was tested on six representative slides from different patients. Autofluorescence was totally quenched by incubation with haematoxylin. No cells were found staining for CD34 but cells staining for CD31 (PECAM-1) were easily located. The latter is effectively the positive control for the technique.



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Fig. 3 (a) Human umbilical vein endothelial cells (HUVECs) recovered from seeding into whole Blood. Note the large number of beads (median perhaps 7.5 beads) rosetted around each HUVEC. (b) One CEC rosetted with 5 beads and two unrosetted cells (that seem likely to be leukocytes) purified from the whole blood of a patient with IRP. (c) A single CEC with at least 9 rosetted beads, prepared from the whole blood of a patient with IRP. (For scale, each bead has a diameter of 4 µM.)

 
Ischaemic rest pain compared to acute myocardial infarction
Levels of CECs in the AMI group were significantly greater (median (IQR) 4.9 (3.6–8.4) cells/mL, ) compared to those in patients with IRP. However, levels of von Willebrand factor (mean/SD 125 [25] IU/dL, ) or tissue factor (median/IQR 24.5 (16–107) ng/mL, ) were not different between the groups.

Discussion

Endothelial cell damage/dysfunction, an accepted component of the pathophysiology of cardiovascular disease, can be assessed by changes in endothelial cell responses to altered blood flow (e.g. flow mediated dilatation) and differences in levels of endothelial cell specific molecules in the blood (e.g. von Willebrand factor).1,4 More recently, damage to the (presumably coronary) vasculature has also been defined as the presence of increased numbers of CECs.12 Increased numbers of immunologically defined CECs in patients with sickle cell disease, systemic lupus erythematosus, vasculitis, thalassaemia and cancer have also been taken to imply gross damage to the endothelium.18–23 We suggest that these cells are not angioblasts as they fail to express CD34.14

Our primary finding of increased numbers of CECs in patients with severe PAD (i.e. with IRP) compared to patients with stable arterial disease (i.e. IC) to some extent reflects our previous finding in coronary artery disease where we found high levels of CECs in severe disease (i.e. AMI) but no increase in more stable (and less concerning) disease (i.e. stable angina). However, higher levels of CECs in our patients with AMI compared to IRP suggest that the former produces a more injurious effect on the endothelium. This may be because, in AMI, there may be endothelial cell detachment associated with plaque rupture. This was not present in controls or those with effort angina because of the lack of this mechanism.4,12 The present study, conducted in the same manner but on a parallel group of patients with PAD, comes to broadly similar conclusions. As the two patient groups (IRP and IC) were roughly matched for the risk factors for atherosclerosis, these seem unlikely account for differences in CECs, although we were not adequately powered to fully test this hypothesis. Sinzinger et al.24 have previously reported increased levels of morphologically defined CECs in peripheral vascular disease.

Our secondary finding was a strong correlation between numbers of CECs and the levels of von Willebrand factor, a good plasma marker of endothelial cell damage/dysfunction5,6 that predicts adverse cardiovascular events.7 This important link between two different methods for assessing endothelial integrity essentially supports the hypothesis that endothelial cell damage is important in cardiovascular disease.1,2 Notably, George et al.22 also reported a crude correlation between increased CECs and von Willebrand factor in infection with Rickettsia; both indicators also resolved during successful treatment. Our third major finding was a less strong, but still significant, correlation between CECs and tissue factor. Raised plasma levels of the latter, produced by monocytes, macrophages and endothelial cells, have been described in cardiovascular disease.9–11 It is still too early to say whether or not raised plasma tissue factor carries a poor prognosis, but such raised levels may contribute to a heightened risk of thrombosis in patients with severe atherosclerosis. Interestingly, tissue factor has been proposed as a marker of endothelial cell injury25 and the significant correlation with von Willebrand factor in this study, as in our previous report,10 may support this concept, although doubts regarding specificity remain. Furthermore, CEC’s from patients with sickle cell disease express increased tissue factor antigen.26

Although it is not widely accepted that IRP due to atherosclerosis of the peripheral arteries is associated with plaque rupture, but more likely with progressive disease leading to thrombosis,3,4 increased numbers of CECs are still present. Despite the much larger amount of (leg) tissue that is ischaemic in IRP compared to the coronary endothelium in AMI, the number of CECs present in the AMI group were significantly higher, a result not reflected in the plasma von Willebrand factor levels. As it is generally accepted that rising von Willebrand factor levels reflect disease severity and progression in both peripheral and coronary artery disease,1,6,7 this may be in keeping with the hypothesis that the pathogenesis of AMI and IRP are different.

Although our present study is limited by its cross-sectional design, we are adequately powered to show differences between the groups studied. In addition, we have confirmed previous observations of raised CECs in patients with AMI,12 and demonstrated for the first time, evidence of direct endothelial cell injury in patients with PAD who have severe disease, i.e. IRP. We have also shown that these patients demonstrate abnormal plasma markers related to endothelial injury, i.e., von Willebrand factor, alongside a weaker, but still significant correlation with pro-coagulant tissue factor, that the number of CECs significantly correlate with plasma von Willebrand factor levels, and that CD146-defined CECs seem unlikely to be CD34-defined angioblasts. Thus, raised CECs in both peripheral and coronary atherosclerotic vascular disease reflect the greatest degree of unequivocal endothelial cell injury.

Acknowledgments

Natali Chung is supported by a non-promotional research fellowship from Merck Sharpe and Dohme. We acknowledge the support of the City Hospital Research and Development Programme for the Haemostasis Thrombosis and Vascular Biology Unit. Immunostaining for CD34 and CD31 was made possible with help from Jennifer Stoddard and Glyn Woodward.

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