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Increased low-density lipoprotein (LDL)-cholesterol is a risk factor for atherosclerosis--a disease in which damage to the endothelium is believed to be an important early step. Increased levels of the endothelial marker von Willebrand factor (vWF) in the plasma of patients with hypercholesterolaemia and atherosclerosis probably reflect this process. In this study we seek to link the established observation that oxidised LDL-cholesterol is cytotoxic to human umbilical vein endothelial cells (HUVECs) in vitro with the common finding of raised plasma vWF in patients with atherosclerosis by incubating HUVECs with physiological/pathological levels of native and oxidised LDL-cholesterol for up to 48 h. Microphotography revealed morphological changes in the HUVECs within 24 h, becoming severe at 48 h, which was mirrored by increased levels of vWF (ELISA) and the release of preloaded radioactive (111)indium tracer into culture supernatants. Our data support and extend the hypothesis that oxidised LDL is directly cytotoxic to HUVECs, and, in addition, provide an important link between in vitro studies and clinical studies where endothelial cell markers such as vWF are increased in the plasma of patients with hypercholesterolaemia and atherosclerosis.

Separate reports have identified differences in plasma levels of the endothelial markers soluble E-selectin, von Willebrand factor (vWf) and soluble thrombomodulin in each of the major modifiable risk factors for atherosclerosis (smoking, hypertension and hypercholesterolaemia), and abnormal levels of some plasma markers predict various adverse cardiovascular events. However, it is unclear whether there is an increasing effect on the endothelium with a worsening risk-factor profile. We measured the three endothelial cell markers by enzyme-linked immunosorbent assay in the plasma of 200 subjects (mean age, 54 years; 58% men) free of the symptoms and clinical signs of atherosclerosis. Levels of the markers were then correlated with the Framingham coronary heart disease (CHD) and cerebrovascular disease (CVD) scores to help determine which (if any) may be useful as good laboratory predictors of future cardiovascular events in prospective epidemiological studies. vWf correlated with CHD (r(s) = 0.269, < 0.001) and CVD risk (r(s) = 0.331, P < 0.001), but soluble E-selectin correlated only with CHD risk (r(s) = 0.163, P = 0.021). We conclude that, of the three specific endothelial markers, vWf correlates most closely with the Framingham risk-factor prediction scores and therefore may be the better plasma endothelial marker of the future development of an atherothrombotic event.

Peripheral artery angiography, a common diagnostic procedure, may cause early and late adverse reactions, such as anaphylaxis, thrombosis and possible progression of the underlying arterial disease. To test the hypothesis that radiographic contrast medium may contribute to these events by adversely affecting the endothelium, leucocytes and/or platelets, 19 subjects undergoing angiography for the investigation and/or treatment of lower limb atherosclerosis were recruited. Blood was obtained from the external iliac vein before, and at serial intervals after, the injection of radiographic contrast medium into the ipsilateral femoral artery for diagnostic use. Markers of endothelial cell injury (von Willebrand factor (vWf)), platelet activation (soluble P-selectin) and leucocyte activation (neutrophil elastase and soluble L-selectin) were measured in citrated plasma. Soluble intercellular adhesion molecule-1 (sICAM-1) and thromboxane B(2), which are non-specific markers of inflammation, were also measured. Compared with the sample prior to angiography, levels of soluble L-selectin and sICAM-1 were reduced (p<0.02) immediately after passage of the last bolus of contrast medium. 15 min later, levels returned to normal but the level of vWf had increased (p<0.02). After 30 min, only levels of thromboxane B(2) were increased (p<0.05). The following day both vWf (p<0.01) and soluble P-selectin (p<0.05) were increased. These data point to both early and late effects of contrast medium on markers of endothelial, platelet and leucocyte function.

Damage to the endothelium is an important component of atherosclerosis and can be quantified by measuring plasma markers, such as von Willebrand factor, thrombomodulin, intercellular adhesion molecule-1, and E-selectin. We hypothesized that increased levels of these markers would be related to objectively defined disease severity among patients with peripheral atherosclerosis or carotid atherosclerosis. To test this, we measured the markers by using ELISA in the plasma of 45 patients with intermittent claudication alone and in 53 patients presenting with transient ischemic attack. Disease severity in the former was by ankle-brachial pressure index and in the latter by ultrasound defined % stenosis. Any symptomatic dual disease or history or present coronary atherosclerosis warranted exclusion. Data were correlated according to Spearman's method. The only significant correlation was between von Willebrand factor and ankle-brachial pressure index (r = -0.39, p = 0.008). Our data suggest that von Willebrand factor is the most sensitive marker of peripheral atherosclerosis and that none of the plasma markers seems to be a useful marker of the degree of carotid artery stenosis.

Cigarette smoking is a risk factor for the development of atherosclerosis. Possible mechanisms for this include leucocytes and platelet activation, and/or damage to the endothelium, any of which may contribute to changes in thrombosis and haemostasis. We examined the acute effects of smoking on these systems by obtaining blood before, immediately after, and at 10 and 30 min after the rapid smoking of two cigarettes in sequence by 20 smokers. Blood samples taken at the same time points from ten non-smokers acted as control material. In the smokers there was a transient rise in leucocyte count and neutrophil activation, but von Willebrand factor (VWF--marking endothelial damage) increased steadily at each time point (P <0.05). There were no changes in neutrophil elastase, soluble intercellular adhesion molecule-1 (sICAM-1 normally increased in smokers), fibrinogen, platelet count or soluble P-selectin (marking platelet activation, also normally increased in smokers). We conclude that the acute smoking of two cigarettes in succession will activate leucocytes and cause endothelial cell damage, but will not immediately influence platelet activity.

Development of an abdominal aortic aneurysm (AAA) may be a product of generalised atherosclerosis. If that is indeed the case, we would expect similarities in various risk factors and other markers in common with occlusive peripheral arterial disease (peripheral arterial disease), and less congruity with healthy controls. To test this hypothesis, we recorded the major risk factors for atherosclerosis, two markers of endothelial dysfunction, and soluble adhesion molecules in 21 patients with an uncomplicated AAA free of symptomatic peripheral arterial disease, 42 patients with peripheral arterial disease, and 42 healthy controls who were matched, as a group, for age and sex. After adjusting for smoking, there were no significant differences in blood pressure, fibrinogen, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1 or lipoproteins between the groups. However, markers of endothelial integrity von Willebrand factor and soluble thrombomodulin were both higher (P < 0.05) only in peripheral arterial disease patients. Relative to the controls, platelet marker soluble P-selectin was increased in AAA (P < 0.01) and in the peripheral arterial disease patients (P < 0.05). Levels were higher in AAA patients than in peripheral arterial disease patients (P < 0.05). Our laboratory data suggest that the pathophysiology AAA and peripheral arterial disease are not identical.

We examined the relationship between specific endothelial cell markers soluble E-selectin, von Willebrand factor and soluble thrombomodulin and the location or extent of atherosclerosis by analysing plasma samples from 200 patients with symptomatic peripheral vascular disease and 213 age- and sex-matched asymptomatic control subjects. Using ELISAS, we found increased von Willebrand factor and thrombomodulin (both P < 0.0001) in the patients relative to the control subjects, but no significant change in soluble E-selectin. Soluble thrombomodulin was increased in patients with disease at one locus (i.e. of the carotid or iliac/femoral arteries), with an additional significant increase in patients with disease at multiple loci (i.e. any combination of carotid, coronary or iliac/femoral artery disease). No marker differentiated carotid artery disease from iliac/femoral artery disease. We conclude that von Willebrand factor is a marker of generalized atherosclerosis, but that soluble thrombomodulin is related to the extent of disease. Further research into these endothelial cell products are warranted to explore their diagnostic and/or prognostic potential.

von Willebrand factor and soluble thrombomodulin are established plasma markers of endothelial cell dysfunction, whilst beta thromboglobulin is an established plasma marker of platelet activity. Soluble P-selectin may be the product of either or both types of cell and levels of all four molecules have been previously found to be increased in atherosclerosis. To determine the relationship of soluble P-selectin to the endothelial cell and platelet products, we measured the four indices in a case control study of 55 patients with peripheral vascular disease and 55 age and sex matched controls. von Willebrand factor (p < 0.0001), beta thromboglobulin (p = 0.0006), soluble P-selectin (p = 0.0021) and soluble thrombomodulin (p = 0.021) were all raised in the patients. Soluble P-selectin correlated with beta thromboglobulin (r = 0.34, p = 0.019) but failed to correlate with either endothelial cell marker. Co-culture of endothelial cells in vitro with bovine thrombin resulted in increased levels of von Willebrand factor in the supernatants but levels of soluble thrombomodulin and soluble P-selectin were not enhanced. Exposure of endothelial cell monolayers to elastase resulted in different patterns of release of von Willebrand factor, soluble thrombomodulin and soluble P-selectin. We suggest that soluble P-selectin is unlikely to arise from the endothelium and may be a new marker of platelet activation in atherosclerosis.

Our objective was to determine whether endothelial cell products von Willebrand factor and soluble E-selectin are related to serum lipids, lipoprotein (a), and vascular disease in patients with hyperlipidaemia. In order to achieve our aim, blood samples were obtained for four experiments from 1) 160 patients (49 with symptomatic vascular disease) with hypercholesterolaemia and an equal number of age and sex matched controls; 2) 31 patients who were studied serially before and after successful resolution of their hypercholesterolaemia; 3) 15 patients with hypertriglyceridaemia; and 4) 20 controls, half of whom consumed a lipid-rich breakfast. von Willebrand factor and soluble E-selectin were measured by enzyme linked immunosorbent assay (ELISA) using commercial reagents. In experiment (1) von Willebrand factor was increased in the patients with hypercholesterolaemia (P=0.0077) and was higher still in patients with vascular disease (P<0.0001). Soluble E-selectin was not influenced by hypercholesterolaemia or vascular disease. The correlation of von Willebrand factor with total and LDL cholesterol (both P<0.001) remained after both age and blood pressure were controlled. Experiment (2) showed that serial studies in patients over an average of 7 months a reduction in total cholesterol was associated with a reduction in von Willebrand factor (r=0.51, P=0.002). Experiment (3) demonstrated that von Willebrand factor was not increased in patients with hypertriglyceridaemia (median 8.9 mmol/L), and in experiment (4) a lipid-rich breakfast taken by fasted, healthy controls produced an increase in serum triglycerides (P<0.01) but did not influence von Willebrand factor over an 8 hour period. We conclude that von Willebrand factor, but not soluble E-selectin, is raised in hypercholesterolaemia and therefore may be a potential indicator of endothelial cell physiology in subjects with, or at risk of, atherosclerotic vascular disease.

A major constraint in vascular surgery is the failure of the currently available prosthetic grafts to match the patency of autogenous vein below the inguinal ligament. Endothelial seeding aims to produce an endothelial lining which is otherwise absent from prosthetics. This could improve patency by preventing thrombosis and the release of mitogens by activated platelets implicated in the development of neointimal hyperplasia. Experimental evidence supports the potential of the technique but clinical trials have been less conclusive. A number of problems need to be solved if this application of cell biology is to become a part of vascular surgical practice.

von Willebrand factor (vWf) is an interesting and potentially important molecule whose biology in health and disease warrants attention. A growing body of knowledge now suggests that plasma levels of this specific product of the endothelial cell may have potential as a marker for the assessment of endothelial injury in vivo. As its functions include platelet aggregation and mediation of platelet adhesion to the subendothelium, it may also have a role in the pathogenesis of progression of atherosclerosis. In comparison to asymptomatic controls, increased levels of vWf are found in atherosclerotic vascular disease and in the presence of several of its major risk factors (smoking, hypercholesterolaemia, hypertension, obesity and diabetes). High plasma levels of vWf are also associated with the prediction of adverse clinical events such as myocardial infarction and poor outcome following arterial surgery, possibly by the promotion of thrombus formation. These and other studies indicate that research directed towards determining whether therapy to reduce levels of vWf also influences the progression of arterial disease should prove to be profitable.

von Willebrand factor (vWf), risk factors for atherosclerosis, body mass index (BMI) and waist-to-hip ratio (WHR) were measured in 108 non-diabetic patients attending lipid and vascular disease clinics and in 107 normal asymptomatic controls. High levels of vWf and increased BMI relative to controls were found in patients with hyperlipidaemia and vascular disease, but WHR was higher only in patients with vascular disease. Total serum cholesterol concentration (P < 0.001), systolic blood pressure (P < 0.001), smoking (P < 0.02) and BMI (P < 0.001), but not WHR, were associated with vWf. As raised levels of vWf are a probable indicator of endothelial damage in vascular disease, these data suggest that obesity has an adverse influence on the endothelium and may help explain its link with cardiovascular disease.

The effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p < 0.01), the smoking markers cotinine (p < 0.02) and thiocyanate (p < 0.01), and was more cytotoxic to endothelial cells in vitro (p < 0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p < 0.001) and cytotoxicity of serum to endothelial cells in vitro (p < 0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p < 0.02), number of years smoking (r = 0.41, p < 0.001) and cotinine (r = 0.45, p < 0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

The relationship between von Willebrand factor antigen (vWFAg, a specific endothelial cell product) and hypertension was examined. Circulating vWFAg levels were measured in serum from patients attending a hypertension clinic and in normotensive controls. The vWFAg was higher in those patients with uncontrolled hypertension at 131 +/- 34 IU/dl than in controls (90 +/- 30 IU/dl) and in those whose hypertension was controlled 104 +/- 29 IU/dl (P < 0.001). Levels of vWFAg correlated with both SBP (r = 0.42, P < 0.0002) and DBP (r = 0.25, P < 0.05), but serum from neither group of patients was more cytotoxic to cultured endothelial cells in vitro than was serum from controls. Neither symptoms of cardiovascular or peripheral vascular disease or two of its risk factors (hypercholesterolaemia or smoking, alone or in combination) appeared to further increase vWFAg in patients with uncontrolled hypertension. However, vascular disease and the same risk factors did increase vWFAg to 133 +/- 36 IU/dl in those patients with controlled hypertension (P < 0.001). These data indicate a relationship between vWFAg and hypertension and suggest that endothelial damage may indeed be important in the vascular complications of hypertension.