Potential biomarkers for vascular damage in ALzheimer's disease: Thrombomodulin and von Willebrand factor

Abstract

Objectives

Evidence regarding the vascular basis of Alzheimer's disease (AD) is growing. In vascular damage thrombomodulin tears of the cell wall and its level increases in the plasma. von Willebrand factor (vWF) is also thought to be a biomarker for vascular damage. The aim of this study was to examine the levels of vWF and thrombomodulin in AD as possible markers for vascular damage and to test their utility as an early biomarker in AD.

Design

Case-control study.

Setting

Geriatric medicine outpatient clinic of a university hospital.

Participants

Twenty Alzheimer's disease patients free from vascular risk factors and 20 controls were enrolled in the study.

Measurements

Thrombomodulin and VWF levels of 20 AD patients and 20 controls were analyzed by commercial kits.

Results

Thrombomodulin levels were not different between Alzheimer's disease and control groups [median (range) = 4.25 (2.27–37.00) ng/ml in Alzheimer's disease and 3.55 (2.27–14.00) in control group, p=0.15]. Von Willebrand Factor antigen (%) levels were 188.5 (96–306) in Alzheimer's disease, and 181 (112–284) in control group (p=0.74).

Conclusion

Although vascular damage is thought to play role in the pathogenesis of AD, vWF and thrombomodulin failed to demonstrate the vascular damage in AD. Their utility to be used as early biomarkers of AD could not be shown.

Introduction

Vascular basis of Alzheimer’s disease (AD) is being studied recently and it has been postulated that vascular risk factors constitutes risk also for AD (1, 2). Endothelial dysfunction, which is an early indicator of atherosclerosis, is a predictor of vascular disease and it has recently been shown to occur in AD patients (3).

Studies concerning the events taking place in the vascular endothelium, which is the dynamic surface of the vascular system, have given clues about the relationship between AD and the endothelium (3, 4, 5). The relationship between AD and the endothelium is a new area of investigation which can elicit important findings about the development, progression, and even prevention of the disease. Some biomarkers indicating endothelial dysfunction and vascular damage have been studied (6, 7). Two of the leading biochemical markers indicating endothelial dysfunction are von Willebrand factor (vWF) and thrombomodulin (7). However, vWF level is also affected by inflammatory processes so it is not specific for vascular damage. On the other hand, thrombomodulin is known to come off the cell surface during endothelial damage resulting in increased serum levels (7). Therefore, increased levels of thrombomodulin can be considered a specific indicator of vascular damage.

The aim of this study was to examine the levels of vWF and thrombomodulin in AD as possible markers for vascular damage occurring in this disease and to test their utility as an early biomarker in AD.

Materials and methods

Subjects and Cognitive Assessment

Forty elderly subjects free from vascular risk factors (20 AD and 20 controls) were enrolled in the study. Exclusion criteria were diabetes mellitus (history of diabetes mellitus, antidiabetic medication, or fasting plasma glucose>126mg/dl), hypertension (blood pressure>140/90mmHg), dyslipidemia (total cholesterol>200mg/dL), evident stroke, documented coronary artery disease (stable angina pectoris, unstable angina pectoris, myocardial infarction, positive result in treadmill test or thallium scanning, coronary intervention, documented coronary artery disease by coronary angiography), history of myocardial infarction, heart failure, peripheral artery disease, acute or chronic infection, malignancy, renal disease, rheumatologic diseases, alcohol abuse, smoking (patients who were ex-smokers for at least 10 years were not excluded), and drugs that may affect endothelial function.

Both groups underwent comprehensive geriatric assessment and neuropsychiatric assessment including Mini-Mental State Examination (MMSE) test (8) and clock drawing test (9). After cognitive assessment and neuroimaging performed by magnetic resonance (MR) imaging, Alzheimer’s disease diagnosis was made according to DSM-IV (Diagnostic and Statistical Manual of Mental Disorders) (10) and NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer Disease and Related Disorders Association) criteria (11). Clinical Dementia Rating Scale (CDR) was used to determine the severity of dementia (12).

The study was approved by the local ethics committee and its protocol was consistent with the declaration of Helsinki. Informed consent was obtained from each patient before enrollment.

Laboratory Assessment

Blood samples were obtained by venipuncture of antecubital vein after an overnight fast. Venous blood samples were drawn into 3.8% trisodium citrate (1:9 dilutions) containing tubes without venous occlusion. Thrombomodulin levels were measured by a microenzyme immunoassay technique using commercially available kits (Immunoassay of thrombomodulin Diagnostica Stago, Asnieres-Sur-Seine, France). The von Willebrand factor antigen was studied by the commercial kit (STA-Liatest vWF; Diagnostica Stago, Asniéres, France).

Statistical Analysis

Categorical variables are demonstrated as percentages. All continuous variables had skew distribution, so they are presented as medians (range). In order to make comparison between AD and control groups, Mann-Whitney U-test was used for skew distributed continuous variables and chi-square test was used for comparing categorical variables. Thrombomodulin and vWF levels were compared between AD and control groups by Mann Whitney U test.

A p value ≤0.05 was considered as statistically significant. Statistical package SPSS 11.0 for Windows was used.

Results

Median (range) ages were 77.5 (67–85) in the Alzheimer’s disease group (female/male 9/11) and 74.5 (65–82) in the control group (female/male 7/13). Age and gender distributions were not significantly different between groups. General characteristics and demographic properties of the study population are given in Table 1.

Table 1.

Demographic properties and general characteristics of the study population

	AD (n=20)	Control (n=20)	p
Age
(median [range])	77.5 (67–85)	74.5 (65–82)	NS
Gender F/M	9/11	7/13	NS
MMSE
(median [range])	19.5 (3–23)	28 (24–30)	<0.001
Thrombomodulin (ng/ml)
(median [min-max])	4.25 (2.27-37.00)	3.55 (2.27-14.00)	NS
Von Willebrand Factor	188.5 (96.0-306.0)	181.0 (112.0-284.0)	NS
antigen (%) (median [range])

AD: Alzheimer’s disease, F/M: female/male, MMSE: Mini-Mental Status Examination Test

Mann Whitney U test revealed that thrombomodulin levels were not different between Alzheimer’s disease and control groups [median (range) = 4.25, (2.27-37.00) ng/ml in Alzheimer’s disease and 3.55 (2.27-14.00) in control group, p=0.15]. Von Willebrand Factor antigen (%) levels were 188.5 (96–306) in Alzheimer’s disease, and 181 (112–284) in control group (p=0.74). Although there was a trend for higher values in AD, the differences did not reach the statistical significance. Thrombomodulin and vWF levels according to groups are shown in Figures 1 and 2.

Figure 1.

Distribution of thrombomodulin levels according to Alzheimer’s disease (AD) and control groups. The box plot shows thrombomodulin values for AD and control groups. The black lines within the boxes indicate the median, the edges of the boxes are the 25th and 75th percentiles, and the lines extend to the maximum and minimum values. Mean values ± SD of thrombomodulin (ng/ml) of AD vs. control group were 4.25 (2.27-37.00), vs. 3.55 (2.27-14.00), respectively; p=0.15

Figure 2.

Distribution of von Willebrand Factor antigen levels according to Alzheimer’s disease (AD) and control groups. The box plot shows thrombomodulin values for AD and control groups. The black lines within the boxes indicate the median, the edges of the boxes are the 25th and 75th percentiles, and the lines extend to the maximum and minimum values. Mean values ± SD of von Willebrand Factor antigen (%) of AD vs. control group were 188.5 (96–306), vs. 181 (112–284), respectively; p=0.74

Discussion

This study showed that thrombomodulin and vWF levels were not affected by Alzheimer’s disease (AD). Although AD was reported to be related to endothelial dysfunction and these parameters are possible markers for endothelial dysfunction, we failed to show their utility as an early biomarker of AD.

The endothelium is the monolayer of endothelial cells lining the lumen of the vascular beds and is mechanically and metabolically strategically located, separating the vascular wall from the circulation and the blood components (13). Endothelial dysfunction is the first step of atherosclerosis and is thought to be impaired in AD as well as many other vascular diseases (3). It was put forward that the toxic effect of amyloid beta may affect endothelial cells and cause endothelial-dependent vasoconstriction (4, 14). Endothelial alterations in patients with AD was detected by measuring the response to acetylcholine iontophoresed into the skin using laser Doppler flowmetry by Khalil et al. (5). Dede et al. demonstrated impaired endothelial function in AD by using a noninvasive method of flow mediated dilation (FMD) (3). It can be hypothesized that beta-amyloid protein may be leading to lipid peroxidation and reactive oxygen species which results in impaired vascular endothelium. Biomarkers that influence the integrity of the vascular endothelium may be important in detecting early vascular injury thus may be a marker for early AD.

There are certain molecules whose elevated levels indicate endothelial damage. Thrombomodulin, endothelial selectin (E-selectin), endothelial leukocyte adhesion molecules, beta thromboglobulin, vWF antigen, fibrinogen, prothrombin fragment, and anticardiolipin antibodies are some of these markers (6, 7). Within these molecules most appropriate ones for laboratory analysis are von Willebrand Factor antigen, thrombomodulin, tissue plasminogen activator level, and soluble E-selectin. Serum soluble intercellular adhesion molecule-1, soluble E-selectin, and thrombomodulin were evaluated as potential markers for endothelial dysfunction in different studies, and significantly greater levels were detected in subjects with AD (6, 7).

Von Willebrand Factor (vWF) antigen is a prothrombotic molecule released from endothelial cells when they are injured or stimulated by some factors (7). It was found to be associated with ischemic heart disease and peripheral artery disease (15, 16). Since vascular damage is thought to play role in the pathogenesis of AD, relationship between vWF and AD was being studied, but the present study did not demonstrate a significant relationship. However, vWF is associated not only with vascular events, but also with inflammatory processes. Therefore, it can not be regarded as a specific marker for endothelial dysfunction. Borroni et al. also failed to find a relationship between vWF and AD which supports results of the present study (7).

Thrombomodulin is an endothelial cell receptor which is a marker of endothelial and platelet activation. In case of endothelial injury it tears of the cell wall and its level increases in the plasma. It shows endothelial damage independent from inflammatory cytokines (7). Blann et al indicated that raised soluble thrombomodulin is a new marker for the progression of atherosclerosis in patients with ischemic heart disease (17). In the present study, thrombomodulin was evaluated as a possible early marker for atherosclerosis occurring in AD. However, no significant relationship could be determined between thrombomodulin levels and AD.

Conclusion

In this study, levels of vWF and thrombomodulin did not differ between AD and control groups. Although vascular damage is thought to play role in the pathogenesis of AD, vWF and thrombomodulin failed to demonstrate the vascular damage in AD. Further studies with larger number of patients and other possible markers are needed to find a biomarker for early diagnosis of AD.