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Cardiovascular Research 2003 57(2):563-571; doi:10.1016/S0008-6363(02)00699-5
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Copyright © 2003, European Society of Cardiology

Vitamin E inhibits lipid peroxidation-induced adhesion molecule expression in endothelial cells and decreases soluble cell adhesion molecules in healthy subjects

Bastiaan van Dama,d, Victor W.M van Hinsberghd,e, Coen D.A Stehouwera,d, Amanda Versteilenb,d, Henk Dekkerc, Rien Buytenheke, Hans M Princene and Casper G Schalkwijkb,d,*

aDepartment of Internal Medicine, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
bDepartment of Clinical Chemistry, Institute for Cardiovascular Research, Vrije Universiteit Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands
cDepartment of Oncology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
dInstitute for Cardiovascular Research, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
eGaubius Laboratory TNO-PG, Leiden, The Netherlands

c.schalkwijk{at}vumc.nl

* Corresponding author. Tel.: +31-20-444-3680; fax: +31-20-444-3895.

Received 21 May 2002; accepted 23 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: In a combination of in vivo and in vitro studies, we investigated mechanisms via which {alpha}-tocopherol, a lipid soluble form of vitamin E, can directly affect endothelial activation as induced by H2O2 and TNF{alpha}. Methods: We measured effects of {alpha}-tocopherol on H2O2-induced lipid peroxidation as determined with a fluorescent C-11 BODIPY581/591 probe and on adhesion molecule expression in cultured endothelial cells. In 20 healthy volunteers treated with increasing doses of {alpha}-tocopherol up to 800 IU/ml for 12 weeks, plasma levels of soluble cell adhesion molecules (sCAMs) and C-reactive protein were measured. Results: We showed that {alpha}-tocopherol protects cultured endothelial cells against H2O2-induced lipid peroxidation, while TNF{alpha} did not induce lipid peroxidation. Moreover, {alpha}-tocopherol attenuated H2O2-, but not TNF{alpha}-induced increases in adhesion molecule expression. In healthy persons, {alpha}-tocopherol decreased plasma levels of sE-selectin from 65±6 to 60±6 ng/ml (P=0.002), sVCAM from 893±31 to 853±23 ng/ml (P=0.022), and sICAM from 483±21 to 463±16 ng/ml (P=0.048). C-Reactive protein, as a sensitive marker of low grade inflammation, was not significantly affected. Conclusion: {alpha}-Tocopherol specifically inhibits lipid peroxidation-induced endothelial activation in vitro. The observed vitamin E-induced decrease in sCAMs in control subjects suggests that lipid peroxidation can take place in healthy individuals. Although vitamin E supplementation may be especially effective in specific groups of patients exposed to increased oxidative stress, our study suggests that vitamin E supplementation can be of benefit in healthy individuals as well.

KEYWORDS Endothelial function; Free radicals; Lipid metabolism


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Oxidative stress produced during inflammatory responses has been implicated as a cause of damage to the vasculature [1,2] and may play an important role in the development of many diseases including atherosclerosis [3]. Due to their location, endothelial cells are continuously exposed to oxidants derived from extracellular sources, mainly superoxide and hydrogen peroxide (H2O2) [4]. In addition, these oxidants are produced intracellularly upon activation by cytokines, including tumor necrosis factor-{alpha} (TNF{alpha}), and can function as second messengers [5].

Activation of endothelial cells by oxidants may lead to a wide range of functional changes such as an increased expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin, and the production of chemokines, such as monocyte chemoattractant peptide-1. The resulting attraction and transendothelial migration of monocytes are believed to be critical to the initiation and progression of atherosclerosis [6,7].

TNF{alpha}-induced endothelial activation leads to translocation of the transcription factor nuclear factor (NF-){kappa}B [8,9], which can be inhibited by antioxidants [10,11], indicating reactive oxygen species as second messengers. However, it has been shown that endothelial cells and other cells respond differently to H2O2 and TNF{alpha} [12]. The mechanisms that explain the difference between H2O2- and TNF{alpha}-induced endothelial activation are unclear. True et al. showed, in human dermal microvascular endothelial cells, that both TNF{alpha} and H2O2 can induce translocation of NF{kappa}B to the nucleus and its binding to DNA, whereas TNF{alpha} can generate additional signals that induce phosphorylation and transcriptional activation of the bound NF{kappa}B p65 [13]. Others showed that H2O2 alone cannot activate NF{kappa}B in endothelial cells [14]. It has also been shown that H2O2 activates ICAM-1 transcription via elements within the ICAM-1 promotor that are distinct from NF{kappa}B-mediated expression induced by TNF{alpha} [15].

Epidemiological studies have demonstrated an association between increased intake of the antioxidant vitamin E and reduced morbidity and mortality from coronary artery disease [16–19]. The protective effects of vitamin E may be related to its ability to decrease levels of soluble adhesion molecules [20]. The mechanisms involved are incompletely understood. It is largely believed that vitamin E serves a protective function by preventing the oxidation of LDL [21,22] or by attenuating effects of ox-LDL [20,23]. However, it has been difficult to link the potential antiatherogenic action of vitamin E to its ability to increase the resistance of LDL to oxidation [24–26]. Vitamin E can also directly affect endothelial activation induced by several inducers of reactive oxygen species (ROS) [27]. Whether the effect of vitamin E on endothelial cells is related to inhibition of lipid peroxidation is unknown.

Therefore, in a combination of in vitro and in vivo studies, we investigated mechanisms via which {alpha}-tocopherol, a lipid-soluble form of vitamin E, can directly affect endothelial activation as induced by H2O2 and TNF{alpha}. Firstly, we measured the effects of {alpha}-tocopherol on H2O2- and TNF{alpha}-induced lipid peroxidation in cultured endothelial cells with the oxidative sensitive fluorescent C-11 BODIPY581/591 probe [28]. This fluorophore is readily incorporated into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid, thereby losing its bright red fluorescence with a shift to green. It is relatively insensitive to nitric oxide and superoxide [28]. Secondly, we compared the effects of {alpha}-tocopherol on H2O2-induced and TNF{alpha}-induced adhesion molecule expression. Thirdly, we assessed, in a group of healthy volunteers, the effects of {alpha}-tocopherol on plasma levels of soluble adhesion molecules, i.e., putative markers of in vivo expression of adhesion molecules.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Materials
D,L-{alpha}-Tocopherol was obtained from Calbiochem (La Jolla, CA, USA). C-11 BODIPY581/591 probe was obtained from Molecular Probes (Junction City, OR, USA). All other compounds were purchased from Sigma (St. Louis, MO, USA).

2.2 Cell culture
Human endothelial cells from umbilical vein (HUVEC) were isolated, cultured and characterized as described previously [29]. Confluent monolayers of endothelial cells between passages 2–3 were used for experiments.

2.3 Oxidative stress measurement
Endothelial cells were cultured in a 96-well plate and pre-incubated overnight in M199 supplemented with 10% (v/v) human serum and 10 ng/ml bFGF with or without {alpha}-tocopherol, and/or L-buthionine-[S,R]-sulfoximine (BSO). The H2O2-induced oxidative stress was assessed with a fluorescence assay using the fluorophore C11-BODIPY581/591 as described [28]. Briefly, cells were incubated for 30 min at 37 °C with C11-BODIPY581/591 (1 µM). Subsequently, the cells were washed with PBS and oxidative stress was induced in PBS supplemented with 5 mmol/l glucose and 0.04, 0.1 or 0.2 mmol/l hydrogen peroxide. A Tecan multiwell spectrofluorometer (Tecan, UK) was used to measure the oxidation of C11-BODIPY581/591. The red fluorescence was selectively detected using the excitation and emission bandpass filters of 590/15 and 635/20 nm, respectively. Under these conditions, increases in H2O2-induced oxidative stress were accompanied with a linear decrease in fluorescence (Fig. 1A). Oxidative stress measurements were performed in triplicate and repeated every 2 min during 1 h. Oxidative stress was expressed as oxidative stress coefficient, being equivalent to the slope of this linear reduction in fluorescence over time.


Figure 1
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  		Fig. 1 Effects of {alpha}-tocopherol on H2O2-induced lipid peroxidation in HUVECs. (A) After incubation with C11-BODIPY581/591, HUVECs were exposed to H2O2 (0.04 or 0.2 mmol/l). The figure shows the mean values of fluorescence as a percentage of the initial value measured in triplicate and the slope of the decrease in fluorescence. (B) Effect of different concentrations of {alpha}-tocopherol on H2O2-induced lipid peroxidation. Confluent endothelial cells were exposed for 18 h to medium supplemented with vitamin E. Data are the mean±S.E.M. of three independent experiments. P for difference of vitamin E versus control: 6.25 µmol/l, P=0.783; 25 µmol/l, P=0.019; 100 µmol/l, P=0.008. (C) Endothelial cells were exposed for 18 h to culture medium supplemented with either {alpha}-tocopherol (25 µmol/l), BSO (1 mmol/l), or a combination of both and thereafter oxidative stress was induced with H2O2 and lipid peroxidation was measured. Data are the mean±S.E.M. of three independent experiments. (D) Serial images of HUVECs examined by confocal laser scanning microscopy and exposed for 30 min to 0.1 mmol/l H2O2 (a). In (b–d) cells were pre-incubated for 48 h prior to exposure to the oxidants with {alpha}-tocopherol (100 µmol/l), BSO (1 mmol/l) or a combination of both, respectively.

 
Glutathione was determined by a HPLC method as described by Neuschwander-Tetri and Roll [41].

2.4 Confocal laser scanning microscopy
For fluorescence microscopy, cells were plated 2 days before the start of the experiment on gelatin-coated coverslips (diameter 24 mm). Samples were placed in a temperature (37 °C)-controlled coverslip holder and images were taken with a Leica TCS4D confocal laser scanning system on an inverted microscope (Leica Microsystems, Heidelberg, Germany), with an argon–krypton laser as excitation source. The green and red fluorescence of C11-BODIPY was acquired simultaneously using double wavelength excitation (laserlines 488 and 568 nm) and detection (emission bandpass filters 530/30 and 590/30).

2.5 EC surface expression of leukocyte adhesion proteins
After an overnight pre-incubation with or without {alpha}-tocopherol, cells were washed with PBS, supplemented with 5 mmol/l glucose and 0.1% human serum albumin. Oxidative stress was induced with 0.1 mmol/l H2O2 for 15 min. Thereafter, PBS was replaced by M199 supplemented with 10% (v/v) human serum and 10 ng/ml bFGF with or without 10 U/ml TNF{alpha} (kindly provided by Dr. J. Tarvernier, Gent, Belgium). The expression of cell-bound VCAM-1, ICAM-1 and E-selectin was measured after 3, 6 and 24 h in 96-well tissue culture plates with a cell-bound ELISA as described in detail [30]. In this procedure, cells are fixed with a low concentration of glutaraldehyde (0.025%) and the cell-bound expression of the adhesion molecules is detected with specific monoclonal antibodies. No dissociation of the cells from their matrix is required avoiding any risk of proteolysis of adhesion molecules. Since the cells are not permeabilised, the adhesion molecule expressed on the cellular membrane is detected. Furthermore, Adhesion molecules were measured in quadruplicate and expressed in arbitrary units (AU), 1 AU being equivalent to 1% of the extinction at 450 nm measured after a stimulation with 10 U/ml TNF{alpha} during 6 h.

2.6 Measurement of soluble adhesion molecules and C-reactive protein (CRP) after vitamin E supplementation in a group of healthy volunteers
Twenty healthy non-smoking volunteers (10 men and 10 women, aged 21–31 years) ingested {alpha}-tocopherol as previously described [21]. Briefly, 25, 50, 100, 200, 400 and 800 IU/day D,L-{alpha}-tocopherol acetate was ingested consecutively during six 2-week periods. Concentrations of soluble cell adhesion molecules (sCAMs) and C-reactive protein (CRP) were assessed in plasma samples taken at baseline and at the end of the study. We used ELISAs (Diaclone SAS, Besancon, France) to measure sE-selectin, sVCAM-1 and sICAM-1. For comparability with other studies, it should be noted that in the group of healthy adults the plasma levels of sICAM-1 was 483 ng/ml sICAM-1 when assayed by the Diaclone sICAM-1 kit and 248 ng/ml when assayed by the R&D sICAM-1 kit. CRP was assayed with a homemade sensitive ELISA, with a lower limit of detection of 0.01 mg/l [31].

The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.7 Statistical evaluation
Data are presented as mean±S.E.M. from three to six experiments. We performed MANOVA (general linear model for repeated measures) to ascertain the significance of differences among groups after different time-intervals. We then used paired t-tests to compare observations where appropriate. The level of significance was set at 5%.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effects of {alpha}-tocopherol on H2O2- and TNF{alpha}-induced oxidative stress in cultured endothelial cells
Stimulation of C11-BODIPY581/591-loaded endothelial cells with increasing concentrations of H2O2 (0.04–0.2 mmol/l) caused a time- and concentration-dependent oxidation of the fluorophore. The H2O2-induced oxidation of C11-BODIPY581/591 was partially inhibited by a preincubation of endothelial cells overnight with {alpha}-tocopherol at a concentration of 25 µmol/l, while a pretreatment with 100 µM of {alpha}-tocopherol completely prevented the oxidation of C11-BODIPY581/591 (Fig. 1). Preincubation of endothelial cells with BSO (1 mmol/l) for 24 h reduced the glutathione levels from 0.44±0.05 to 0.005±0.002 µmol/µg protein (n=4, P=0.005). Preincubation of the cells with the same concentration of BSO for 48 h significantly enhanced the H2O2 (0.04 and 0.2 mmol/l)-induced oxidation of C11-BODIPY581/591, as determined by an increased oxidative stress coefficient (from 0.10±0.02 to 0.79±0.01 AU (n=3, P=0.002) and from 0.48±0.08 to 0.78±0.05 AU (n=3, P=0.033), respectively). When these cells were also pretreated with {alpha}-tocopherol (25 or 100 µmol/l), the effect of BSO on C11-BODIPY581/591 oxidation was completely abolished (oxidative stress coefficients induced by 0.04 and 0.2 mmol/l H2O2 were 0.12±0.02 and 0.25±0.03 AU, respectively) (Fig. 1). In contrast to H2O2, no effect of TNF{alpha} on the oxidation of C11-BODIPY was observed (data not shown).

3.2 Effects of {alpha}-tocopherol on H2O2-induced adhesion molecule expression in cultured endothelial cells
We next determined if the lipid peroxidation induced by H2O2 was accompanied by endothelial activation as measured by an increased expression of adhesion molecules. H2O2 increased both the expression of E-selectin and VCAM-1, but not ICAM-1 (Fig. 2). Six hours after exposure of endothelial cells to H2O2 (0.1 mmol/l, 15 min), VCAM-1 expression was significantly increased from 19±2 to 30±2 AU (n=6, P=0.002), and E-selectin expression was increased from 19±1 to 63±4 AU (n=3, P=0.008). Twenty-four hours after exposure of endothelial cells to H2O2, the expression of adhesion molecules had returned to control values. If H2O2-induced lipid peroxidation is involved, pretreatment with the antioxidant {alpha}-tocopherol may have beneficial effects. Indeed, pretreatment with {alpha}-tocopherol (25 or 100 µmol/l), significantly inhibited not only the H2O2 (0.1 mmol/l)-induced lipid peroxidation (Fig. 1), but also the H2O2-induced increased expression of VCAM-1 and E-selectin (Fig. 2). While basal expression of ICAM-1 was not significantly increased by H2O2, preincubation with {alpha}-tocopherol led to slightly, but significantly lower levels of ICAM-1 6 h after exposure of endothelial cells to H2O2 (83±4.1 vs. 74±3.1 AU, n=5, P=0.008). In contrast to the effect of TNF{alpha} (see below), we were not able to detect shed adhesion molecules sVCAM-1, sE-selectin and sICAM-1 in the supernatants of the cultured endothelial cells with or without H2O2 (data not shown). This is probably due to the fact that the values remain below the detection limit of the ELISA.


Figure 2
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  		Fig. 2 Effects of {alpha}-tocopherol on H2O2-induced adhesion molecule expression in HUVECs. Expression of adhesion molecules in controls (open circles), cells exposed to hydrogen peroxide (white squares), and cells exposed to hydrogen peroxide after preincubation with 25 µmol/l {alpha}-tocopherol (gray squares) or 100 µmol/l {alpha}-tocopherol (black triangles). Data are the mean±S.E.M. of three to six independent experiments. Shown in the figure are P values lower than 0.05 for significance of differences for control versus H2O2 (*), H2O2 versus H2O2+vitamin E 25 µmol/l (#) and H2O2 versus H2O2+vitamin E 100 µmol/l (+).

 
3.3 Effects of {alpha}-tocopherol on TNF{alpha}-induced adhesion molecule expression in cultured endothelial cells
As shown in Fig. 3, TNF{alpha} caused a sustained increase in both VCAM-1 and ICAM-1 expression. E-selectin expression was increased after exposure to TNF{alpha} during 3 and 6 h, but not after 24 h. Although TNF{alpha} had no effect on lipid peroxidation as measured by the oxidation of C11-BODIPY581/591, we assessed whether {alpha}-tocopherol was able to affect the TNF{alpha}-induced expression of adhesion molecules. Pretreatment for 18 h with {alpha}-tocopherol (25 µmol/l) did not attenuate effects of TNF{alpha} on adhesion molecule expression (Fig. 3). Furthermore, {alpha}-tocopherol did also not affect the TNF{alpha}-induced increased levels of sE-selectin, sICAM and sVCAM as detected in the supernatants (data not shown). Similar results were found at a concentration of 100 µmol/l vitamin E (data not shown). These data indicate that lipid peroxidation is not involved in the TNF{alpha}-induced expression of these adhesion molecules.


Figure 3
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  		Fig. 3 Effects of {alpha}-tocopherol on TNF{alpha}-induced adhesion molecule expression in HUVECs. Expression of adhesion molecules in controls (open circles), cells exposed to TNF{alpha} (black triangles), and cells exposed to hydrogen peroxide after preincubation with 25 µmol/l {alpha}-tocopherol (gray squares). Data are the mean±S.E.M. of three independent experiments. Shown in the figure are P values lower than 0.05 for significance of differences for control versus TNF{alpha} (*), and TNF{alpha} versus TNF{alpha}+vitamin E 25 µmol/l (#).

 
3.4 Effects of {alpha}-tocopherol on soluble cell adhesion molecules (sCAMs) and CRP in healthy volunteers
To evaluate whether vitamin E affects endothelial activation in vivo, we assessed the effects of supplementation of vitamin E on sE-selectin, sVCAM-1 and sICAM-1 in healthy adults. At the end of a period of 12 weeks, in which a group of healthy volunteers (n=20) was treated with increasing doses of {alpha}-tocopherol up to 800 IU/ml, all sCAMs were shown to be decreased as compared with the start of the study. sE-selectin was decreased from 65±6 to 60±6 ng/ml (P=0.002), sVCAM from 893±31 to 853±23 ng/ml (P=0.022), and sICAM from 483±21 to 463±16 ng/ml (P=0.048). Plasma levels of CRP were not significantly affected by treatment with {alpha}-tocopherol (Fig. 4). Overall, CRP levels increased non-significantly from 1.68±0.6 to 2.08±1.3 mg/l (P=0.568).


Figure 4
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  		Fig. 4 Effects of vitamin E on plasma sCAMs and CRP levels in healthy volunteers. Plasma levels of sE-selectin, sVCAM-1, sICAM-1 and CRP of healthy volunteers before and after treatment with vitamin E for 12 weeks. The data are expressed as box-and-whisker plots. The central line is the median, the lower and upper quartiles are indicated by the box and the 2.5 and 97.5 centiles are indicated by whiskers.

 
If values from one subject with CRP-levels exceeding 10 mg/l at the start of the study were excluded, changes in CRP-levels remained non-significant (change from 1.18±0.29 to 0.81±0.24 mg/l, P=0.164). Changes in sE-selectin (from 66±6 to 60±6 ng/ml, P=0.001) and sVCAM-1 (from 902±30 to 859±23 ng/ml, P=0.016) remained significant, while sICAM-1 was no longer significantly affected by vitamin E (change from 483±22 to 462±16 ng/ml (P=0.053).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we report that stimulation of endothelial cells with H2O2, but not TNF{alpha}, led to lipid peroxidation, as measured with the oxidative sensitive C11-BODIPY581/591 probe, and that pretreatment with {alpha}-tocopherol dose-dependently inhibited this H2O2-induced lipid peroxidation. Moreover, we showed that {alpha}-tocopherol was able to attenuate H2O2-induced increases in adhesion molecule expression, but did not affect TNF{alpha}-induced endothelial activation. In healthy volunteers, we found that {alpha}-tocopherol decreased sCAMs. Vitamin E had no significant effect on CRP, a sensitive marker of low-grade inflammation.

Increased oxidative stress leading to endothelial activation has been implicated in the increased cardiovascular risk of diabetes mellitus and hypertension [32,33]. Important sources of H2O2 are tissue granulocytes [34], neutrophils adherent to vascular cells [4], and xanthine oxidase, which can be generated in response to ischemia–reperfusion injury [35]. After diffusion across cell membranes, H2O2 is converted into O2•– or hydroxyl radicals (OH•) via the Haber–Weiss reaction. Our results show that stimulation of cultured endothelial cells with H2O2 is associated with oxidation of C11-BODIPY581/591, indicative of increased lipid peroxidation in the cell membrane [28].

In cultured endothelial cells, it has been demonstrated that vitamin E is taken up in a time- and dose-dependent manner without causing toxicity in a concentration up to 100 µM [36]. We demonstrated that vitamin E, in the same concentration range, dose-dependently attenuated H2O2-induced lipid peroxidation (Fig. 1). Several mechanisms may account for this effect of vitamin E. It can react with radicals such as the lipid peroxyl radical and may play a role in the inhibition of lipid peroxidation in cellular membranes. Furthermore, it has been proposed that vitamin E mainly degrades H2O2 by increasing the intracellular level of glutathione [37]. We cannot exclude that part of the effects of vitamin E observed in this study are mediated via this latter mechanism. However, we also showed that vitamin E effectively inhibits the conversion of BODIPY581/591 by H2O2 if glutathione is completely depleted by BSO, indicating that vitamin E directly reacts in the lipid peroxidation reaction as a free radical chain reaction breaker.

We showed that 100 µmol/l of H2O2, besides its effects on lipid peroxidation, led to an increased endothelial expression of E-selectin and VCAM-1, but not ICAM-1 (Fig. 2). Based on the experiments of Roebuck et al., that demonstrated a H2O2-induced increase in ICAM-1 gene transcription via AP-1/Ets elements within the ICAM-1 promoter [42], increased ICAM-1 protein expression might be expected. However, no H2O2-induced expression of ICAM-1 was detected in our study, which may indicate impaired translation or instability of mRNA. Furthermore, we cannot rule out that, due to high constitutive expression of ICAM-1 and detection techniques used for the detection of ICAM-1, we have missed minimal changes in the expression patterns.

The levels of H2O2 used in this study are comparable with levels that are released by tissue-resident leukocytes [38]. Moreover, a 5-min pulse of 100 µmol/l of H2O2 is capable of inducing leukocyte rolling in postcapillary venules of Sprague–Dawley rats [39]. The effects of H2O2 on adhesion molecule expression could be prevented by vitamin E (Fig. 2). These findings implicate lipid peroxidation as an alternative pathway via which H2O2 activates endothelial cells.

We found no effect of TNF{alpha} on lipid peroxidation. Although TNF{alpha}-induced VCAM-1 and E-selectin expression can be inhibited by the hydrophilic antioxidants PDTC [10] and NAC [11], vitamin E did not affect TNF{alpha}-induced adhesion molecule expression in this study (Fig. 3). One explanation for this finding is that the lipid-soluble {alpha}-tocopherol, being mainly localised in the cellular membranes [36], may not be able to reach intracellular compartments where ROS are formed upon TNF{alpha}-induced activation of endothelial cells. Indeed, it has been shown that the hydrophilic {alpha}-tocopheryl succinate can suppress TNF{alpha}-induced NF{kappa}B mobilization [40]. Thus, it appears that lipid peroxidation in the cellular membrane does not play a role in TNF{alpha}-induced E-selectin, VCAM-1 and ICAM-1 expression, whereas the H2O2-induced expression of E-selectin and VCAM-1 is dependent on lipid peroxidation.

In line with the observation that vitamin E decreased H2O2-induced E-selectin and VCAM-1 expression, but did not affect TNF{alpha}-induced endothelial activation in vitro, we found, for the first time, that 800 IU/day vitamin E decreased sE-selectin and sVCAM-1 in healthy volunteers. It did not affect CRP, a sensitive marker of cytokine-induced inflammation. This suggests that under our experimental conditions, beneficial effects of vitamin E on soluble adhesion molecules are primarily caused by its ability to reduce lipid peroxidation within the membranes of endothelial cells and probably not by a reduction of low-grade inflammation. However, it has been reported that more intensive treatment with 1200 U/ml vitamin E for 3 months is able to reduce sCAMs, which is in agreement with our study, but also the release of the proinflammatory cytokines IL-1β and TNF{alpha} in healthy subjects and diabetic patients [20].

The decrease in sICAM-1 levels after vitamin E supplementation in vivo contrasts with the absence of effects of vitamin E on either H2O2- or TNF{alpha}-induced ICAM-1 expression in vitro. Possibly, H2O2 can induce ICAM-1-expression only in the presence of additional activating factors that are present in vivo, but not in vitro. Indeed, it has been shown that H2O2 alone can increase NF{kappa}B-translocation without increasing ICAM-1 expression, due to an absent phosphorylation of the bound NF{kappa}B subunit p65, which is necessary for activating the ICAM-1 promotor [13]. Furthermore, the decrease in sICAM expression after vitamin E supplementation was less pronounced than the decreases in sE-selectin and sVCAM (Fig. 4).

Although several epidemiological studies have shown that increased dietary vitamin E intake is linked to a decreased risk of atherosclerotic events [16–19], controlled clinical trials present contradictory results [43–45]. Part of this discrepancy may be caused by the fact that presently it not possible to directly quantitate oxidative stress in vivo and hence it is difficult to select patients likely to benefit from vitamin E supplementation and to calculate an effective dose of vitamin E. Indirect measures of oxidative stress in vivo include the measurement of isoprostanes as indices of lipid peroxidation [46] and an ex vivo assay of the oxidizability of LDL cholesterol [47]. Contradictory results have been obtained by these methods with regard to effects of vitamin E on oxidative stress in healthy individuals. The decrease in oxidizibility of LDL after supplementation of vitamin E in this population [21] is in line with a study of Jialal et al. [48]. Moreover, Davi et al. [49] and Marangon et al. [50] showed that vitamin E can decrease isoprostane levels. However, these results contrast with a recent report showing no effects of vitamin E supplementation on isoprostane levels [51]. Measurement of sCAMs may provide an attractive alternative to define optimal antioxidant regimens for specific populations before initiating large-scale clinical trials. Although vitamin E supplementation may be especially effective in specific groups of patients exposed to increased oxidative stress, such as hypertensive diabetic patients [33] or patients undergoing conditions involving ischemia–reperfusion [35], our study suggests that vitamin E supplementation may be of benefit in healthy individuals as well.

Time for primary review 27 days.


   Acknowledgements
 
Rebecca Gallimore, Jan van Bezu and Harry Twaalfhoven are acknowledged for technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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