Vitamin E Supplementation Reduces Plasma Vascular Cell Adhesion ...

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The Journal of Clinical Endocrinology & Metabolism 87(6):2940 –2945 Copyright © 2002 by The Endocrine Society

Vitamin E Supplementation Reduces Plasma Vascular Cell Adhesion Molecule-1 and von Willebrand Factor Levels and Increases Nitric Oxide Concentrations in Hypercholesterolemic Patients GIOVAMBATTISTA DESIDERI, MARIA CONTINA MARINUCCI, GIANLUCA TOMASSONI, PIER GIORGIO MASCI, ANNA SANTUCCI, AND CLAUDIO FERRI University of L’Aquila, Department of Internal Medicine and Public Health, 67100 Coppito–L’Aquila, Italy Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and reduced nitric oxide (NO) availability represent early characteristics of atherosclerosis. To evaluate whether the antioxidant vitamin E affected the circulating levels of soluble VCAM-1 (sVCAM-1) and the plasma metabolite of NO (nitriteⴙnitrate) in hypercholesterolemic patients, either vitamin E (either 400 IU or 800 IU/d for 8 wk) or placebo were randomly, double-blindly given to 36 hypercholesterolemic patients and 22 age- and sexmatched controls. At baseline hypercholesterolemic patients showed higher plasma sVCAM-1 (␮g䡠literⴚ1) (591.2 ⴞ 132.5 vs. 505.0 ⴞ 65.6, P < 0.007) and lower NO metabolite (␮M) levels (15.9 ⴞ 3.4 vs. 29.2 ⴞ 5.1, P < 0.0001) than controls. In hypercholesterolemic patients, 8 wk vitamin E (but not placebo) treatment significantly decreased circulating sVCAM-1 levels (400 IU:

T

HE UP-REGULATION OF vascular cell adhesion molecule-1 (VCAM-1) allows the interaction of vascular endothelial (1) and smooth muscle (2) cells with integrins ␣4␤1 and ␣4␤7 present on monocytes and lymphocytes and the consequent transendothelial migration of these cells. Thus, VCAM-1 up-regulation represents a fundamental step in the initiating events of atherogenesis (3–5). VCAM-1 expression by human endothelial cells is regulated by an antioxidant-sensitive transcriptional regulatory mechanism acting on nuclear translocation of transcriptional factor nuclear factor KB (6). Although poorly expressed by the resting endothelium, up-regulation of VCAM-1 is rapidly stimulated in vitro by IL-1, TNF-␣, and interferon-␥ (5). Furthermore, modified (7) and oxidized low-density lipoproteins (LDLs) (8) increase VCAM-1 expression. In accord with this, animal models of hypercholesterolemia have demonstrated augmented expression of VCAM-1 associated with atherosclerosis (9). In keeping with this, a cholesterol-enriched diet induced a rapid up-regulation of VCAM-1 in aortic endothelial cells (10). In regard to human data, several studies attempted to identify the possible relationship between dyslipidemia and VCAM-1 by studying the soluble (s) form of the adhesin. This latter is released by activated human vascular endothelial cells (11), as well as by nonendothelial cell sources such as Abbreviations: HDL, High-density lipoprotein; LDL, low-density lipoprotein; NO, nitric oxide; VCAM-1, vascular cell adhesion molecule-1; sVCAM-1, soluble VCAM-1; VLDL, very low-density lipoprotein; vWf, von Willebrand factor.

ⴚ148.9 ⴞ 84.6, P < 0.009; 800 IU: ⴚ204.0 ⴞ 75.7, P < 0.0001; placebo: ⴚ4.7 ⴞ 22.6, NS), whereas it increased NO metabolite concentrations (400 IU: ⴙ4.0 ⴞ 1.7, P < 0.02; 800 IU: ⴙ5.5 ⴞ 0.8, P < 0.0001; placebo: ⴙ0.1 ⴞ 1.1, NS) without affecting circulating lowdensity lipoprotein levels. Changes in both plasma sVCAM-1 and NO metabolite levels showed a trend to significantly correlate (r ⴝ ⴚ0.515, P ⴝ 0.010; and r ⴝ 0.435, P ⴝ 0.034, respectively) with changes in vitamin E concentrations induced by vitamin E supplementation. In conclusion, isolated hypercholesterolemia both increased circulating sVCAM-1 and reduced NO metabolite concentrations. Vitamin E supplementation counteracts these alterations, thus representing a potential tool for endothelial protection in hypercholesterolemic patients. (J Clin Endocrinol Metab 87: 2940 –2945, 2002)

vascular smooth muscle cells (2), and can be detected in the serum (12), thus representing an index of VCAM-1 expression by the vascular endothelium (12). By this approach, Hackman et al. (13) failed to find significantly raised sVCAM-1 levels in hypercholesterolemic patients. Similar findings were observed in patients with familial hypercholesterolemia (14). On the other hand, a previous report by our group (15) showed significant correlation between sVCAM-1 and LDL cholesterol in hypercholesterolemic patients, thus suggesting that LDL might affect VCAM-1 expression in vivo. In conflict with this hypothesis, 51% reduction of serum LDL levels, attributable to aggressive lipid-lowering therapy, has been reported to exert limited effects on plasma sVCAM-1 concentrations (13); whereas two different antioxidants, i.e. vitamin E and probucol, were able to prevent oxidized LDL-induced VCAM-1 up-regulation in vitro (8). These data suggest that reduced peroxidation of native LDL in the vascular wall, rather than decrement of circulating LDL concentrations, might affect VCAM-1 expression in vivo. Taken together, the above data clearly indicate that convincing answers to the following two key questions are still lacking: 1) whether hypercholesterolemia per se might promote endothelial activation; and 2) whether antioxidant treatment might counteract such endothelial activation in hypercholesteromic patients. The aim of this study was to give definite replies to the above two questions. For this purpose, we evaluated circulating levels of sVCAM-1 and von Willebrand factor (vWf), the latter representing a well-established in vivo marker of endothelial cell

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activation and damage (16, 17), in patients with isolated hypercholesterolemia. Moreover, because endothelial nitric oxide (NO) production is tonically involved in the inhibition of VCAM-1 expression (18, 19), we also evaluated the relationship between sVCAM-1 and NO metabolite (nitrite⫹nitrate) levels in our study population. Finally, we evaluated the effect of treatment with two different doses of vitamin E on circulating sVCAM-1, vWf, and NO metabolite levels in patients with elevated serum LDL concentrations. Materials and Methods Thirty-six never-treated hypercholesterolemic patients (20 males and 16 females; mean age, 47.1 ⫾ 7.5 yr) were recruited in our outpatient unit, on the basis of serum LDL cholesterol levels more than 5.2 mm and less than 7.8 mm and triglyceride levels less than 1.7 mm after 30 d on an American Heart Association step I diet. To exclude influential factors, patients were selected according to the following exclusion criteria: age less than 25 or more than 55 yr, pregnancy, concomitant diseases, personal history of previous cerebro- or cardiovascular diseases, diabetes of either type I or type II (20), hypertension (sitting systolic/diastolic blood pressure levels ⱖ 140/90 mm Hg at four different visits performed at 1-wk interval), obesity (body mass index more than 26 kg䡠m⫺2), smoking, drug consumption (including vitamins, aspirin, birth control pills, and others), alcohol intake more than 10 g/d, proteinuria (i.e. urinary albumin ⬎ 200 ␮g/min), serum creatinine more than 100 ␮m, or atherosclerotic lesions of the neck and limb vessels (as evaluated by clinical and ultrasound studies). Accurate medical history and physical examination, including fundoscopic evaluation, further excluded the presence of clinically evident atherosclerotic lesions of the coronary, cerebral, and peripheral beds. In addition, normal M-mode and B-mode echocardiograms and 12-lead electrocardiogram were requested as inclusion criteria. Patients with allergic diathesis regarding both type I and type II immune responses and/or reporting respiratory, gastrointestinal, or genitourinary tract infections during the last 3 months were also screened out. A group of 22 healthy subjects (12 males and 10 females; mean age, 44.8 ⫾ 7.7 yr), selected according to the above criteria but with serum LDL cholesterol less than 4.1 mm, served as control.

Baseline assessment of sVCAM-1 and NO Both hypercholesterolemic patients and control subjects continued the above diet and were invited to return in our Outpatient Unit, after 12 h at fast, at 0800 h. Then, blood samples for measurements of serum lipid and plasma sVCAM-1, plasma metabolites of NO, vWf, and vitamin E levels were drawn from an antecubital vein, 1 h after an iv catheter was inserted and kept patent by saline infusion (0.2 ml/min). Heparin was not used because of its influence on endothelial adhesion molecule expression (21). Serum lipids were assessed on the same morning of venipuncture, after blood centrifugation. For sVCAM-1, NO metabolite, vWf, and vitamin E measurements, blood samples were centrifuged (after venipuncture) at 2000 ⫻ g and 4 C; plasma was aliquoted into polypropylene tubes and kept frozen at ⫺80 C until assayed.

Vitamin E treatment of hypercholesterolemic patients and control subjects After baseline blood sampling, both hypercholesterolemic patients and control subjects were randomly, double-blindly assigned to receive vitamin E at 400 IU/d (11 patients and 7 controls) or vitamin E at 800 IU/d (13 patients and 7 controls) or placebo (12 patients and 8 controls) over a period of 8 wk. Diet was not changed. Vitamin E was supplied as ␣-tocopherol (Roche Molecular Biochemicals, Milan, Italy) capsules (1 per day) ingested with meal. Blood samplings for measurements of serum lipid and plasma sVCAM-1, NO metabolite, vWf, and vitamin E concentrations were repeated after 4 and 8 wk on both placebo and active treatments. The treatment phase of this study was followed by a skilled researcher of our staff, who was unaware of study design, results, and purpose. This study was approved by our Institutional Ethics Committee. All subjects gave their written informed consent.

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Laboratory methods Serum total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels were assessed by enzymatic methods (Boehringer Ingelheim GmbH, Mannheim, Germany); in the case of HDL cholesterol, after precipitation of LDL and very low-density lipoprotein (VLDL) cholesterol fractions by phosphotungstate. LDL and VLDL cholesterol levels were assessed by the Friedewald method (22). Circulating sVCAM-1 concentrations were assessed by immunoenzymatic methods (R & D Systems, Minneapolis, MN). The assessment of plasma vWf was performed by an immunoenzymatic method (Roche Molecular Biochemicals). Plasma NO metabolite levels were evaluated with measurements of nitrite⫹nitrate by colorimetric detection of nitrite after conversion of all the sample nitrate into nitrite (Assay Design Inc., Ann Arbor, MI). Plasma vitamin E concentrations were measured by HPLC according to the well-established method of Lee et al. (23). Because the absolute vitamin E levels may depend on that of concurrent plasma lipids (24), plasma vitamin E levels were expressed both in absolute concentrations and as the ratio of vitamin E/(total cholesterol⫹ triglycerides), as described (25). Measurements of plasma sVCAM-1, NO metabolite, vWf, and vitamin E concentrations were performed in duplicate. All the above laboratory procedures were performed by technicians who were unaware of the study design, purpose, and results.

Statistical analysis All data were stored on a common database and analyzed by software from SPSS, Inc., Chicago, IL. Differences among the groups were tested for significance by one-way ANOVA followed by the Bonferroni’s test. The paired t test was used to compare intragroup lipid, VCAM-1, vWf, NO metabolite, and vitamin E levels before and after treatment. Linear regression and correlation were used to evaluate relationships between variables. Statistical significance was considered as a P value less than 0.05. Unless otherwise stated, all data are presented as mean ⫾ sd.

Results Baseline evaluation

The general characteristics of the study populations are shown in Table 1. As expected, hypercholesterolemic patients, compared with controls, had higher total and LDL cholesterol and lower HDL cholesterol concentrations. According to the selection criteria, the two groups manifested similar triglyceride, glucose, HbA1c, systolic and diastolic blood pressure levels, and body mass indexes (Table 1). Absolute plasma vitamin E levels were significantly higher in hypercholesterolemic patients than in control subjects (P ⬍ 0.0001), whereas plasma TABLE 1. General characteristics of the study population

No. Sex (m/f) Age (yr) Body mass index (kg䡠m⫺2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Insulin (pM) Glucose (mM) Total cholesterol (mM) LDL cholesterol (mM) HDL cholesterol (mM) VLDL cholesterol (mM) Triglycerides (mM) Vitamin E (␮M) Vitamin E/(total cholesterol ⫹ triglycerides) a

P ⬍ 0.0001 vs. controls.

Patients

Controls

36 20/16 47.1 ⫾ 7.5 24.8 ⫾ 0.8 126.0 ⫾ 6.1 79.0 ⫾ 3.9 86.5 ⫾ 15.4 4.9 ⫾ 0.5 7.7 ⫾ 0.4a 6.2 ⫾ 0.4a 0.9 ⫾ 0.1a 0.6 ⫾ 0.1 1.4 ⫾ 0.1 42.5 ⫾ 10.0a 4.7 ⫾ 1.0

22 12/10 44.8 ⫾ 7.7 24.6 ⫾ 0.9 124.2 ⫾ 7.7 77.9 ⫾ 3.8 81.2 ⫾ 10.5 4.9 ⫾ 0.3 3.6 ⫾ 0.4 2.0 ⫾ 0.3 1.0 ⫾ 0.1 0.6 ⫾ 0.1 1.3 ⫾ 0.1 23.2 ⫾ 5.0 4.6 ⫾ 0.9

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vitamin E concentrations, adjusted for total lipids, were similar in patients and controls (Table 1). As shown in Fig. 1, hypercholesterolemic patients manifested higher circulating sVCAM-1 concentrations (A) and lower plasma NO metabolite levels (B) than normocholesterolemic subjects. Hypercholesterolemic patients also had higher vWf levels, compared with those of normocholesterolemic subjects (Fig. 1C). In hypercholesterolemic patients, circulating sVCAM-1 directly correlated with both LDL cholesterol (r ⫽ 0.583, P ⬍ 0.0001) and vWF (r ⫽ 0.361, P ⫽ 0.031), whereas it inversely correlated with NO metabolite levels (r ⫽ ⫺0.435, P ⫽ 0.008). Plasma vWF directly correlated with LDL cholesterol (r ⫽ 0.394, P ⫽ 0.018), and NO metabolite levels inversely correlated with LDL cholesterol (r ⫽ ⫺0.363, P ⫽ 0.029) in hypercholesterolemic patients. A trend toward a direct relationship between plasma sVCAM-1 levels and LDL cholesterol was evident in normocholesterolemic subjects (r ⫽ 0.415, P ⫽ 0.055). The other metabolic variables (i.e. HbA1c, glucose, total cholesterol, HDL cholesterol, triglyceride, and fasting insulin levels), as well as blood pressure levels and body mass index, did not correlate with plasma sVCAM-1 concentrations.

sVCAM-1 and NO metabolite responses to vitamin E

FIG. 1. Plasma sVCAM-1 (␮g䡠liter⫺1, A), NO metabolite (nitrite⫹nitrate, ␮M, B), and vWf (kU䡠liter⫺1, C) levels in 36 normotensive, nondiabetic, nonobese patients with elevated serum LDL but normal triglyceride levels (open circles) and control subjects (black circles). Squares and vertical bars are mean and SD, respectively.

FIG. 2. Changes in plasma sVCAM-1 (␮g䡠liter⫺1, A) and NO metabolite (nitrite⫹nitrate, ␮M, B) concentrations in normotensive, nondiabetic, nonobese patients with elevated serum LDL but normal triglyceride levels after 8 wk of placebo (n ⫽ 12) or 400 IU/d vitamin E (n ⫽ 11) or 800 IU vitamin E (n ⫽ 13).

Hypercholesterolemic patients and control subjects who were assigned to receive either 400 IU vitamin E or 800 IU vitamin E or placebo treatments were matchable for age, sex, blood pressure levels, and metabolic variables. Treatment of hypercholesterolemic patients and control subjects with vitamin E (either 400 IU/d or 800 IU/d) or placebo did not induce significant changes in metabolic variables, including LDL cholesterol concentrations (data not shown). In hypercholesterolemic patients, vitamin E given for 4 and 8 wk significantly reduced plasma sVCAM-1 levels in a dose- and time-dependent fashion (Fig. 2A). Similarly, circulating vWf levels were significantly reduced by 8 wk vitamin E supplementation in hypercholesterolemic patients on both 400-IU and 800-IU/d treatments (Table 2). With regard to plasma NO metabolite levels, they significantly increased in a time-dependent fashion on both vitamin E doses (Fig. 2B). In control subjects, a slight trend toward a reduction of



TABLE 2. Circulating vWf and vitamin E levels in hypercholesterolemic patients and control subjects at baseline and after 8 wk of either vitamin E (400 or 800 IU 䡠 d⫺1) or placebo administration. Variable

Placebo Baseline

Vitamin E 400 IU 8 wk

Baseline

n ⫽ 12

Patients

Vitamin E 800 IU

8 wk

Baseline

n ⫽ 11

vWf (kU䡠liter⫺1)

1.2 ⫾ 0.3

1.3 ⫾ 0.2

1.3 ⫾ 0.3

0.9 ⫾ 0.3a

Vitamin E (␮M)

40.2 ⫾ 7.1

39.1 ⫾ 7.3

43.6 ⫾ 11.9

73.9 ⫾ 19.7

n⫽8

Controls

1.1 ⫾ 0.2

0.7 ⫾ 0.3b

43.8 ⫾ 11.0

95.2 ⫾ 20.7d n⫽7

0.9 ⫾ 0.1

0.9 ⫾ 0.2

1.0 ⫾ 0.2

0.8 ⫾ 0.2

Vitamin E (␮M)

21.7 ⫾ 4.8

23.2 ⫾ 4.0

22.7 ⫾ 4.8

35.1 ⫾ 5.9

SD.

c

n⫽7

vWf (kU䡠liter⫺1)

Data are shown as mean ⫾ a P ⬍ 0.005. b P ⬍ 0.0002. c P ⬍ 0.0004. d P ⬍ 0.0001. e P ⬍ 0.001.

8 wk

n ⫽ 13

e

1.0 ⫾ 0.1

0.8 ⫾ 0.1

25.4 ⫾ 5.4

50.1 ⫾ 10.2b

Significance values are given vs. baseline.

plasma sVCAM-1 (␮g䡠liter⫺1) (400 IU: ⫺18.3 ⫾ 11.9; 800 IU: ⫺23.3 ⫾ 15.5; placebo: ⫺7.5 ⫾ 24.9) and vWf levels (Table 2) and increment in NO metabolite (␮m) concentrations (400 IU: ⫹1.6 ⫾ 1.7 ␮g/liter; 800 IU: ⫹ 2.1 ⫾ 2.0 ␮g/liter; placebo: ⫺0.7 ⫾ 2.0) was observed after vitamin E (but not placebo) treatment. Eight weeks of vitamin E supplementation was followed by a significant increment in plasma absolute vitamin E levels in both hypercholesterolemic patients and control subjects (Table 2). Pooling together hypercholesterolemic patients taking 400 IU/d and 800 IU/d vitamin E, the changes in plasma sVCAM-1 levels observed after 8 wk showed a trend to inversely correlate with the changes in both the vitamin E/(total cholesterol ⫹ triglycerides) ratio and nitrite⫹nitrate concentrations (Fig. 3, A and B, respectively). Furthermore, changes in plasma nitrite⫹nitrate levels observed after vitamin E administration showed a trend to directly correlate with changes in the plasma vitamin E/(total cholesterol ⫹ triglycerides) ratio (Fig. 3C). Discussion

In this study, we observed increased circulating sVCAM-1 concentrations and reduced plasma NO metabolite levels in adult nonobese, nondiabetic normotensive patients with high serum LDL but normal triglyceride concentrations. The elevated levels of plasma sVCAM-1 directly correlated with serum LDL, whereas it inversely correlated with plasma NO metabolite concentrations. Eight weeks of vitamin E supplementation significantly reduced circulating sVCAM-1 levels and increased plasma NO metabolite concentrations in hypercholesterolemic patients. The first interesting finding of this study is the trend of plasma sVCAM-1 to be higher in hypercholesterolemic patients than in normocholesterolemic subjects. Indeed, increased circulating sVCAM-1 concentration might suggest that an early endothelial activation was present in these patients despite the absence of other cardiovascular risk factors and overt vascular damage. Consistent with this hypothesis and in agreement with previous reports (26 –29), hypercholesterolemic patients also displayed increased levels of vWf factor, a well-established marker of endothelial damage (16, 17).

In our report, we did not investigate the mechanisms leading to increased VCAM-1 levels in hypercholesterolemic patients. However, the direct relationship between circulating sVCAM-1 and LDL concentrations, already described in a previous report by our group (15), strongly suggests that LDL could affect VCAM-1 secretion in hypercholesterolemic patients. In this context, it is interesting to note that a slight trend toward a direct relationship between circulating sVCAM-1 and LDL concentrations was observed in control subjects. These data seems to suggest that LDL from hypercholesterolemic patients is more able than LDL from normocholesterolemic subjects in stimulating sVCAM-1 secretion. In keeping with this, hypercholesterolemia often display a variable degree of lipid peroxidation (29), and it is known that LDL can stimulate VCAM-1 expression when subjected to oxidation, both directly (7, 8) and by modulating endothelial cell response to cytokines such as TNF-␣ (30). Thus, LDL has all the biological potential to induce sVCAM-1 release in vivo. Furthermore, hypercholesterolemic patients also displayed lower HDL cholesterol levels than controls. In this regard, HDLs are believed to have antioxidant effects because of their capacity to remove any lipid oxidation product (31). Because VCAM-1 expression by human endothelial cells is regulated by an antioxidant-sensitive transcriptional regulatory mechanism acting on nuclear translocation of transcriptional factor nuclear factor KB (6), we cannot exclude that reduced HDL levels could contribute to the increment in circulating sVCAM-1 levels observed in hypercholesterolemic patients. Finally, hypercholesterolemic patients showed low plasma NO metabolite levels that were inversely correlated with sVCAM-1 concentrations. Because endothelial NO production is tonically involved in the inhibition of VCAM-1 expression (18, 19), we could speculate that reduced NO availability attributable to enhanced LDL oxidation (32) might contribute to increased sVCAM-1 levels in hypercholesterolemic patients. In contrast to our evidence, Hackman et al. (13) failed to find raised sVCAM-1 levels in patients with elevated LDL concentrations. Similar findings have been observed in Afrikaner patients with familial hypercholesterolemia (14).

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FIG. 3. Relationships between changes in plasma sVCAM-1 (␮g䡠liter⫺1) and changes in plasma vitamin E levels adjusted for total lipid concentrations [␮M䡠(mM)⫺1, A] and NO metabolite concentrations (nitrite⫹nitrate, ␮M, B) induced by 8 wk of vitamin E supplementation (open circles, 400 IU/d; black circles, 800 IU/d) in hypercholesterolemic patients. C, Relationship between changes in plasma NO metabolite concentrations (nitrite⫹nitrate, ␮M) and changes in plasma vitamin E levels adjusted for total lipid concentrations [␮M䡠(mM)⫺1] observed in the same patients after vitamin E administration (open circles, 400 IU/d; black circles, 800 IU/d).

Even more perplexing, a previous report by our group (15) showed a slight elevation of plasma sVCAM-1 levels in essential hypertensive patients with mildly elevated cholesterol and triglyceride levels. However, in those various studies (13–15), selection criteria were completely different from those of the current report. Thus, a number of influential factors, such as hypertriglyceridemia (13, 15), previous pharmacological treatment (13), different diet habits attributable to ethnicity (14), and hypertension (15), might have affected plasma sVCAM-1 levels and led to the lack of its significant increase in such patients. Of note, Blann et al. (33) recently described, in hypercholesterolemic patients, sVCAM-1 levels close to those observed in our hypercholesterolemic population. In particular, in this elegant report, authors failed to find significant difference in sVCAM-1 levels between pa-


tients and controls. In our opinion, this fact was attributable to the selection criteria used for the control population. Indeed, for our study, we selected control subjects without allergies, recent infections, and subclinical atherosclerotic lesions, as documented by ultrasound techniques. However, our control subjects showed rather low LDL cholesterol levels. Thus, we cannot exclude that this fact could have contributed, at least partly, to the difference in sVCAM-1 levels observed between hypercholesterolemic patients and controls. The second interesting finding of our report is the reduction of plasma sVCAM-1 levels observed after vitamin E supplementation in hypercholesterolemic patients. Vitamin E, as well as other antioxidants, are able to inhibit the antioxidant-sensitive control mechanisms involved in VCAM-1 expression in human endothelial cells (8, 34). Thus, we could speculate that vitamin E might have affected plasma sVCAM-1 levels by counteracting enhanced lipid peroxidation in hypercholesterolemic patients (29), i.e. inhibiting oxidized LDL-related stimulation of VCAM-1 expression (7, 8, 30). In accord with this, changes in sVCAM-1 levels observed after vitamin E supplementation were inversely correlated with changes in vitamin E levels. In this context, it is interesting to observe that, although vitamin E administration in the normocholesterolemic group had no significant effect on sVCAM-1 levels, there was a trend in the same direction as in the hypercholesterolemic group. These data might suggest that vitamin E is more able to reduce sVCAM-1 levels in the presence of enhanced oxidative stress, which is typical of hypercholesterolemia (29). In keeping with this, vitamin E has been reported to not affect indices of lipid peroxidation in normocholesterolemic subjects (35). However, vitamin E levels in the hypercholesterolemic patients were absolutely higher than those observed in hypercholesterolemic subjects, at both basal and postadministration time points. Thus, we cannot exclude that the different degree of plasma sVCAM-1 level reduction observed in hypercholesterolemic patients and control subjects could be attributable, at least partly, to achieving a certain plasma vitamin E threshold. Moreover, hypercholesterolemic patients also showed a significant increase in plasma NO metabolite levels after vitamin E administration. Because NO inhibits VCAM-1 expression in human vascular endothelial cells (18, 19), it is intriguing to speculate that increased NO availability, induced by vitamin E administration, could have contributed to the reduction of plasma sVCAM-1 levels in hypercholesterolemic patients. Consistent with this hypothesis, changes in plasma NO metabolite levels showed a trend toward being directly correlated with changes in vitamin E levels and inversely correlated with changes in circulating sVCAM-1 concentrations in pooled hypercholesterolemic patients. Taken together, our data clearly demonstrated that vitamin E counteracts endothelial activation in hypercholesterolemic patients. Consistent with this, vitamin E supplementation also reduced vWf levels, which probably represents the most accurate in vivo marker of endothelial cell activation and damage (16, 17). Moreover, our data are in agreement with the improvement in endothelial function observed in hypercholesterolemic patients after vitamin E supplementation (36, 37). However, VCAM-1 expression as well as re-


duced NO availability represent the earliest steps in atherogenesis (4). Thus, vitamin E is likely more effective in preventing early endothelial damage than in reducing atherosclerosis progression in already developed atherosclerotic plaque. In accord with this, high vitamin E intake has been reported to be associated with a low risk of coronary heart disease in patients free of diagnosed cardiovascular disease (38, 39) but not in patients with already established atherosclerotic lesions (40, 41). In conclusion, the current report represents, to our knowledge, the first one clearly demonstrating that vitamin E supplementation counteracts endothelial cell activation in patients with hypercholesterolemia and without other cardiovascular risk factors and atherosclerotic lesions. Thus, vitamin E could represent a new important tool for endothelial protection in isolated hypercholesterolemia. Acknowledgments Received September 14, 2001. Accepted February 20, 2002. Address all correspondence and requests for reprints to: Giovambattista Desideri, M.D., University of L’Aquila, Department of Internal Medicine and Public Health, Blocco 11, Via Vetoio, 67100 Coppito– L’Aquila, Italy. E-mail: [email protected].

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