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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.

Tissue factor (TF) is one of the major initiators of coagulation and raised plasma levels have been found in various cardiovascular diseases. TF activity is, however, regulated by tissue factor pathway inhibitor (TFPI), and alteration in levels of TF and/or TFPI may thus relate to thrombogenesis and atherogenesis. To investigate possible abnormalities in TF and free TFPI (i.e. unbound to TF) and total TFPI among patients with peripheral artery disease (PAD), we studied 42 patients (mean age 57, 35 men) with objectively proven (by ABPI/Doppler) disease and 42 age- and sex- matched healthy controls. TF, free TFPI and total TFPI were measured in citrated plasma by ELISA. TF was higher in the patients with PAD compared to controls (275+/-122 pg/ml versus 158+/-60, P<0.0001) but levels of total TFPI were lower in the patients (43+/-10 ng/ml versus 50+/-15, P=0.021). There was no significant difference in levels of free TFPI between patients and controls (7.2+/-1.5 ng/ml in controls, 7.5+/-1. 6 among patients, P=0.39). Within the control patients, levels of free and total TFPI were significantly correlated (Spearman r=0.51, P=0.001) but in the patients with PAD this correlation was poor (r=0. 21, P=0.178). We suggest that reduced levels of total TFPI and raised levels of TF may contribute to the process of atherogenesis and the increased risk of thrombosis among patients with cardiovascular disease.

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.

Increased levels of the endothelial markers von Willebrand factor and soluble thrombomodulin have been identified as predictors of the development of adverse cardiovascular events. The purpose of this study was to determine which of these markers is the best predictor of such events. Both markers were measured using enzyme-linked immunosorbent assays in 111 subjects at high risk of cardiovascular disease (50 with peripheral atherosclerosis and 66 who had suffered myocardial infarction). After a mean of 46 months, a follow-up investigation was performed and cardiovascular end-points (myocardial infarction, stroke, measured progression of peripheral atherosclerosis, arterial surgery, etc.) were noted. Multivariate analysis revealed that both markers were independent predictors among the 54 subjects who suffered any one of the cardiovascular end-points (P < 0.01). However, only an increased level of von Willebrand factor predicted the outcome among the 39 subjects who suffered a myocardial infarction, stroke or arterial surgery (P < 0.05). We conclude that von Willebrand factor is a marginally better predictor of cardiovascular events than soluble thrombomodulin.

von Willebrand factor antigen II (vWFAgII) is the 100 kDa propolypeptide of endothelial cell marker von Willebrand factor (vWF). Our aim was to determine the relationship between vWFAgII and mature vWF and an additional endothelial cell marker, soluble thrombomodulin, in atherosclerosis. To do this, we measured levels of all three by enzyme-linked immunosorbent assay in plasma obtained from 24 patients with peripheral vascular disease (PVD), from 25 patients who survived a myocardial infarction [i.e. had ischaemic heart disease (IHD)], and from 47 age- and sex-matched controls. We found raised levels of vWFAgII in PVD (57.3 +/- 15.3 microg/dl; mean +/- standard deviation) and in IHD (53.4 +/- 19.2 microg/dl) compared with the controls (35.7 +/- 12.0 microg/dl; analysis of variance P < 0.001). Raised levels of vWf were found in both groups of patients but raised soluble thrombomodulin was found only in patients with PVD. Levels of vWFAgII correlated with those of vWf (r = 0.45, P < 0.001) but not with soluble thrombomodulin (r = 0.14, P = 0.17), nor any of the major risk factors for atherosclerosis. Our brief study reports raised levels of vWFAgII in atherosclerosis. This may, like that of vWf, be related to endothelial cell damage, although the incomplete correlation between the two implies different metabolic and/or release mechanisms.

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.

Two specific endothelial cell products, von Willebrand factor and soluble E-selectin, were measured together with serum lipids, lipoprotein(a), systolic and diastolic blood pressure (SHP, DBP) in a follow up study of 162 patients attending a dedicated lipid clinic. Patients were further classified by the presence or absence of symptomatic vascular disease and smoking. After a mean of 49 months, 45 patients experienced a cardiovascular event (fatal or nonfatal myocardial infarction, stroke, or arterial surgery) and 11 developed non-cardiovascular diseases, including cancer. In univariate analysis, existing vascular disease (P < 0.01), increased levels of von Willebrand factor (P < 0.0001) and low density lipoprotein cholesterol (P < 0.02), greater age (P < 0.01), and lower levels of soluble E-selectin (P < 0.03) were all predictive of future vascular events. However, in multivariate analysis, only increased von Willebrand factor was predictive (P < 0.001). von Willebrand factor was also higher in patients who developed non-cardiovascular disease relative to those free of disease (P < 0.05). Our data support the hypothesis that increased levels of von Willebrand factor are an indicator of poor prognosis in patients with atherosclerosis or its risk factors.

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.

Soluble P-selectin (CD62P) may arise from platelets, the endothelium, or both, and raised levels are found in those with thrombotic disease and atherosclerosis. To determine whether these increased levels in atherosclerosis are related to hypercholesterolaemia, blood samples were obtained from 86 patients (43 with symptomatic vascular disease) attending a hypercholesterolaemia clinic, and 86 age- and sex-matched controls. Parallel measurement of endothelial cell product von Willebrand factor helped define the origin of sP-selectin. Using ELISAs, soluble P-selectin was higher (median 290 ng/ml, range 80-735, P < 0.05) in patients with vascular disease than in both patients with uncomplicated hypercholesterolaemia (median 210 ng/ml, range 55-550), and controls (median 190 ng/ml, range 48-500). Von Willebrand factor was raised in both patients with uncomplicated hypercholesterolaemia (115 +/- 26 IU/dl, P < 0.05) and patients with hypercholesterolaemia and vascular disease (129 +/- 32 IU/dl, P < 0.02) compared with controls (102 +/- 30 IU/dl). Levels of soluble P-selectin did not correlate with von Willebrand factor, total low-density-lipoprotein (LDL) or high-density-lipoprotein (HDL) cholesterol or triglycerides levels, blood pressure or smoking, but von Willebrand factor correlated with LDL cholesterol (r = 0.42, P < 0.05). We conclude that plasma lipoproteins are not a major influence on levels of soluble P-selectin.

Increased levels of the endothelial markers soluble E-selectin (P = 0.011), soluble thrombomodulin (P < 0.0001) and von Willebrand factor (VWF, P < 0.0001) were found in 116 patients with ischaemic heart disease compared to an equal number of age- and sex-matched asymptomatic controls. In a multivariate analysis of the markers versus the major risk factors for atherosclerosis, VWF correlated with total cholesterol (P = 0.002) and E-selectin with sex (lower in women, P = 0.004) and triglycerides (P = 0.007). The data point to profound differences in the release mechanisms of these three endothelial cell products and suggests that further studies into the roles of these molecules in coronary artery disease are warranted.

Objectives: To test the hypothesis that endothelial cell damage and hypoxia are related to the activity of neutrophil elastase in patients with peripheral atherosclerosis., Design: A cross-sectional serological study in a tertiary referral, University Hospital., Materials: Venous blood was obtained from 22 patients with peripheral vascular disease and an equal number of age and sex matched controls., Methods: Neutrophil elastase and two markers of endothelial cell damage (von Willebrand factor and soluble thrombomodulin) were measured in plasma by ELISA. Hypoxia was measured by percutaneous oxygen (by oximeter) at the dorsum of the foot., Results: Patients had higher von Willebrand factor, higher soluble thrombomodulin and higher elastase but lower percutaneous oxygen (all p < 0.001). In the patient's group, there were significant inverse correlates between von Willebrand factor and percutaneous oxygen (p = 0.004) and between soluble thrombomodulin and percutaneous oxygen (p = 0.011) while elastase correlated positively with soluble thrombomodulin (p = 0.023)., Conclusions: Our data support the hypothesis that release of elastase from activated neutrophils relates to endothelial cell damage. This may contribute to hypoxia and may result in the deterioration in clinically assessed atherosclerosis.

Tissue plasminogen activator antigen (tPA), plasminogen activator inhibitor antigen (PAI-1), soluble P-selectin and von Willebrand factor antigen (vWf) were measured by ELISA in 41 patients with peripheral vascular disease (PVD), 41 with ischaemic heart disease (HD) and in 46 age and sex matched asymptomatic controls. Increased vWf was found in patients with IHD (p = 0.0002) and in patients with PVD (p = 0.0011) relative to the controls but levels did not differ between the two patients groups. Raised tPA found in both PVD (p = 0.0006) and IHD (p = 0.0061) compared to the controls also failed to differentiate the two groups of patients. Soluble P-selectin was also raised in both groups (p = 0.003 in IHD and p = 0.0102 in PVD) with no difference between the groups. There were no differences in levels of PAI-1 between the groups. In the subjects taken as a whole, there were significant Spearman's correlations between tPA and vWf (r = 0.37, p < 0.001), tPA and triglycerides (r = 0.38, p < 0.001), tPA and P-selectin (r = 0.19, p = 0.032), vWf and age (r = 0.25, p = 0.005) and inversely between vWf and HDL (r = -0.25, p = 0.006). These data support the concept that increased levels of tPA may be important in atherosclerosis, and indicate that soluble P-selectin may be useful in further analysis of the role of platelets and the endothelial cell in this disease.

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.