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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:1847-1853, August 2005

Biochemical and Molecular Actions of Nutrients

Vitamin E Increases Production of Vasodilator Prostanoids in Human Aortic Endothelial Cells through Opposing Effects on Cyclooxygenase-2 and Phospholipase A21,2

Dayong Wu3, Liping Liu*, Mohsen Meydani* and Simin Nikbin Meydani

Nutritional Immunology Laboratory and * Vascular Biology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111

3To whom correspondence should be addressed. E-mail: dayong.wu{at}tufts.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Impairment of endothelium-dependent vasodilation is associated with the initiation and development of atherosclerosis. Vasodilator prostanoids constitute a protective mechanism in maintaining normal vasomotor function. In the current study, we determined the effect of in vitro vitamin E supplementation at physiologically relevant concentrations (10–60 µmol/L) on the production of the vasodilator prostanoids prostaglandin I2 (PGI2; prostacyclin) and prostaglandin E2(PGE2) by human aortic endothelial cells (HAECs) as well as its underlying mechanism. Results showed that vitamin E dose dependently (10–40 µmol/L) increased the production of both prostanoids by HAECs. This was associated with a dose-dependent (10–40 µmol/L) upregulation of cytosolic phospholipase A2 (cPLA2) expression and arachidonic acid release. In contrast, vitamin E dose dependently (10–60 µmol/L) inhibited cyclooxygenase (COX) activity but did not affect the expression of either COX-1 or COX-2, indicating that the effect of vitamin E on COX activity was post-translational. Thus, vitamin E had opposing effects on the 2 key enzymes in prostanoid biosynthesis; at the concentrations used in this study, this resulted in a net increase in the production of vasodilator prostanoids. The vitamin E–induced increase in PGI2 and PGE2 production may contribute to its suggested beneficial effect in preserving endothelial function.

KEY WORDS: • vitamin E • human aorta endothelial cells • prostanoids • cyclooxygenase • phospholipase A2

Epidemiologic studies have suggested that increased vitamin E intake is associated with reduced morbidity and mortality from cardiovascular disease (CVD)4 (1–5). The mechanism for this effect of vitamin E has been the subject of continuing investigation. Vitamin E inhibits platelet aggregation, LDL oxidation, monocyte adhesion to endothelial cells, and endothelial expression of adhesion molecules, inflammatory cytokines, and chemokines (5–9), all of which are believed to contribute to atherosclerosis, a hallmark of CVD. One of the key mechanisms underlying the action of vitamin E appears to be its ability to maintain or restore endothelial function.

The endothelium is a complex endocrine and paracrine organ that plays a critical role in regulating vasomotor tone, permeability, platelet aggregation, thrombus generation, leukocyte adhesion and translocation, and smooth muscle cell proliferation and migration. Maintenance of a normal vascular tone is critical for endothelial integrity and homeostasis. Impairment of endothelium-dependent vasodilation is an early sign of atherosclerosis (10), and its assessment was proposed to have a prognostic value for CVD outcome in clinical practice (11–13). An animal study reported that the aortic rings isolated from rats fed a diet deficient in vitamin E showed morphological disruption of the endothelium as well as impaired endothelium-dependent vasodilation (14). In human studies, plasma vitamin E was shown to correlate significantly with coronary endothelium-dependent vasodilation in patients who did not take supplemental vitamin E (15). In addition, oral vitamin E supplementation (300 mg {alpha}-tocopherol acetate/d) for 4 wk improved flow-mediated (endothelium-dependent) vasodilation in patients with high remnant lipoprotein levels (16).

The endothelium produces an array of vasomotion-regulating molecules that act in an endocrine, paracrine, or autocrine fashion to regulate vascular tone. Vasodilator molecules such as nitric oxide and certain prostanoids constitute a protective mechanism in vasomotor function. Prostaglandins I2 (PGI2; prostacyclin) and E2 (PGE2), 2 major cyclooxygenase (COX)-derived vasodilator prostanoids, were shown to counteract the function and/or inhibit the secretion of vasoconstrictors such as thromboxane A2 (TXA2) and endothelin-1 (17) and thus have been suggested to play a role in the prevention of CVD. Vitamin E was shown to affect metabolism of arachidonic acid (AA) and thus prostanoid synthesis, but the nature of its action varies depending on the cell type. Therefore, we hypothesized that the favorable effect of vitamin E on vasomotor function is due in part to its modulation of synthesis of vasodilator prostanoids. In the current study we used human aortic endothelial cells (HAECs) as an in vitro model to define the effect of vitamin E supplementation on production of the vasodilator prostanoids PGI2 and PGE2 and its underlying mechanisms.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Cell culture. HAECs were purchased from Cambrex; cells from passages 4–7 were used in this study. Cells were seeded in 10-cm dishes or 24-well plates (BD Labware) coated with 2% gelatin (Sigma) and were grown in EBM-2 medium containing supplements (EGM-TM2 SingleQuots) and 2% fetal bovine serum (FBS). Cells were cultured for all experiments at 37°C, 5% CO2 in atmosphere, and 95% humidity.

    Vitamin E preparation and supplementation. A stock solution of vitamin E (RRR-{alpha}-tocopherol, gift from Cognis) was prepared at 30 g/L in ethanol and stored at –70°C. To efficiently deliver vitamin E to HAECs, the stock solution was first mixed with FBS at a 1:20 ratio, incubated at 37°C for 30 min, and mixed on a vortex every 10 min. The FBS solution thus generated was further diluted with growth medium containing FBS to obtain work solutions containing different concentrations of RRR-{alpha}-tocopherol. When HAECs grew to 80% confluency, they were incubated in the medium supplemented with RRR-{alpha}-tocopherol at 10, 20, 40, or 60 µmol/L, or a vehicle control. All final cultures contained the same levels of FBS (2%) and ethanol (0.2%). After 20 h, the medium was replaced by a medium containing 10 µg/L interleukin-1ß (IL-1ß) (R & D) to stimulate the cells for different periods of time depending on the measured outcome.

    Vitamin E assay. HAECs were incubated in the medium containing 0, 20, 40, or 60 µmol/L RRR-{alpha}-tocopherol for 20 h. Cells were then washed and collected in PBS. Uptake of RRR-{alpha}-tocopherol by HAECs was determined by HPLC using electrochemical detection as described previously (8).

    Determination of prostanoid production and COX activity. After HAECs were stimulated with IL-1ß for 20 h, the culture supernatants were collected to determine prostanoid concentrations using RIA. PGI2 was measured as its stable hydrolytic product 6-keto-PGF1{alpha}. To determine COX activity, cells were incubated in the presence of exogenous AA (Sigma) at 30 µmol/L for 10 min. At the end of the reaction, aspirin (2 mmol/L; Sigma) was added to stop COX activity and the supernatants were collected to determine the conversion of AA to 6-keto-PGF1{alpha} or PGE2 using RIA. Cells were lysed in 1 mol/L NaOH for total cellular protein analysis using the bicinchoninic acid protein assay kit (Pierce). RIA was conducted as previously described (18).

    AA release assay. HAECs were incubated in 24-well plates in the presence of [3H]-AA (0.05 µCi/well) (PerkinElmer) and different concentrations of RRR-{alpha}-tocopherol for 20 h. Cells were then washed 5 times and stimulated with IL-1ß (10 µg/L) for 5 h. In phospholipase A2 (PLA2) inhibition experiments, a specific cytosolic PLA2 (cPLA2) inhibitor, arachidonyltrifluoromethyl ketone (AACOCF3) (1 µmol/L; Sigma), was added to cell cultures after cells were washed with medium. Plates were centrifuged at 200 x g for 10 min, and the supernatants were collected to determine released [3H]AA. Cells were lysed in 1 mol/L NaOH to measure cell-associated [3H]AA. Radioactivity in the samples was measured by scintillation counting. The percentage of AA release was calculated as follows: released AA/(released AA + cell-associated AA) x 100.

    Western blot analysis for COX-1, COX-2, and cPLA2. HAECs were preincubated with RRR-{alpha}-tocopherol at different concentrations for 20 h and then stimulated with 10 µg/L of IL-1ß for 18 h. Cells were washed twice with cold PBS and then harvested in lysis buffer (50 mmol/L NaCl; 50 mmol/L Tris, pH 7.5; 5 mmol/L EDTA; 0.1% SDS; and 1% NP-40). Total cellular protein was quantified using Bio-Rad protein assay reagent, and 20 µg of protein from each sample was electrophoresed in 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk in TBS containing 0.1% Tween-20 for 1 h, the membrane was incubated with 0.5 mg/L of COX-1, COX-2, or cPLA2 antibodies (all from Santa Cruz Biotechnology) for 1 h. Membranes were rinsed and then incubated with 0.1 mg/L of the corresponding secondary antibodies conjugated with alkaline phosphatase (Santa Cruz) for 1 h. After being rinsed, membranes were incubated in a Chemiluminescent Detection System (Tropix) for 4 min and then exposed to film. Loading across the samples was normalized by reprobing stripped membranes with ß-actin antibody (Sigma). The bands were analyzed densitometrically by ChemiImager (Alpha Innotech).

    Statistical analysis. All results are expressed as means ± SEM. Treatment effects were analyzed by 1-way ANOVA followed by Tukey’s test using SYSTAT 10 statistical software (Systat). Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Vitamin E is incorporated into HAECs. Supplementing culture medium of confluent HAECs with different concentrations of vitamin E for 20 h dose dependently increased (P < 0.05) the cellular levels of {alpha}-tocopherol (Fig. 1).



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  		FIGURE 1 Vitamin E uptake by HAECs after in vitro supplementation. HAECs were incubated in the presence of 0, 20, 40, and 60 µmol/L RRR-{alpha}-tocopherol for 20 h. Incorporation of d-{alpha}-tocopherol by HAECs was determined by HPLC. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05.

 
    Vitamin E promotes PGI2 and PGE2 production by HAECs. Based on our previous observation (8), IL-1ß is an efficient simulator of endothelial cell activation, as indicated by the synthesis of marker molecules such as adhesion molecules and chemokines. In this study, IL-1ß stimulation significantly increased production of both PGI2 (measured as its stable hydrolytic product 6-keto-PGF1{alpha}) and PGE2 (Fig. 2). Vitamin E dose dependently increased (P < 0.05) PGI2 production by 31, 90, 136, and 139% in unstimulated HAECs and by 34, 75, 102, and 50% in IL-1ß–stimulated HAECs treated with 10, 20, 40, and 60 µmol/L vitamin E, respectively (Fig. 2A). Similarly, vitamin E increased (P < 0.05) PGE2 production by 40, 173, 279, and 342% in unstimulated HAECs and by 48, 101, 99, and 77% in IL-1ß–stimulated HAECs treated with 10, 20, 40, and 60 µmol/L vitamin E, respectively (Fig. 2B). The maximum effect of vitamin E was observed at concentrations between 40 and 60 µmol/L in unstimulated cells and between 20 and 40 µmol/L in IL-1ß–stimulated cells for both prostanoids.



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  		FIGURE 2 Effect of vitamin E on PGI2 (A) and PGE2 (B) production in HAECs. HAECs supplemented with different concentrations of RRR-{alpha}-tocopherol were stimulated with IL-1ß (10 µg/L). Supernatants were collected to quantify PGI2 (A) (measured as its stable hydrolytic product 6-keto-PGF1{alpha}) and PGE2 (B) using RIA. Values are means ± SEM, n = 6. Means without a common letter (within lower- or uppercase) differ, P < 0.05.

 
    Vitamin E inhibits COX activity of HAECs. COX is a rate-limiting enzyme in the biosynthesis of prostanoids. In most studies, altered production of prostanoids can be attributed to corresponding changes in COX activity. Thus, to determine the mechanism of vitamin E–induced upregulation of PGI2 and PGE2, we further assessed the effect of vitamin E on COX activity. Despite its enhancement of 6-keto-PGF1{alpha} and PGE2 production, vitamin E dose dependently inhibited (P < 0.05) COX activity, as determined by synthesis of either 6-keto-PGF1{alpha} or PGE2 from exogenously added AA (Fig. 3). When cells were treated with vitamin E at 10, 20, 40, and 60 µmol/L for 20 h, COX activity, as determined by conversion of AA to 6-keto-PGF1{alpha}, was reduced to 85, 59, 44, and 33% of the control level in unstimulated HAECs and to 72, 44, 42, and 25% of the control level in IL-1ß-stimulated HAECs, respectively (Fig. 3A). The results were similar when COX activity was determined by conversion of AA to PGE2 (Fig. 3B).



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  		FIGURE 3 Effect of vitamin E on COX activity in HAECs. HAECs were preincubated with RRR-{alpha}-tocopherol and stimulated by IL-1ß (10 µg/L). COX activity was determined by measuring the conversion of AA to 6-keto-PGF1{alpha} (A) or PGE2 (B). Values are means ± SEM, n = 6. Means without a common letter (within lower- or uppercase) differ, P < 0.05.

 
    Vitamin E enhances AA release from HAECs. Because the vitamin E–induced enhancement of prostanoid synthesis could not be explained by the increase in COX activity, we next investigated whether vitamin E enhances prostanoid production through increasing AA release, another key regulatory component in prostanoid synthesis. Vitamin E increased (P < 0.05) AA release by 95, 168, 208, and 222% in unstimulated HAECs and by 34, 89, 116, and 117% in IL-1ß-stimulated HAECs treated with 10, 20, 40, and 60 µmol/L vitamin E, respectively (Fig. 4). IL-1ß increased (P < 0.05) AA release irrespective of vitamin E supplementation (Fig. 4). The percentage of increase in AA release induced by vitamin E was higher than its inhibition of COX activity.



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  		FIGURE 4 Effect of vitamin E on AA release in HAECs. HAECs were incubated in the presence of [3H]-AA (0.05 µCi/well) and different concentrations of RRR-{alpha}-tocopherol for 20 h and then stimulated with IL-1ß (10 µg/L) for 5 h. Percent AA release was calculated as: released AA/(released AA + cell-associated AA) x 100. Values are means ± SEM, n = 5. Means without a common letter (within lower- or uppercase) differ, P < 0.05.

 
    Vitamin E does not affect COX-1 and COX-2 but increases cPLA2 expression in HAECs. Altered COX activity generally reflects a change in the enzymes’ protein level and/or in their catalytic activity. In this study, COX activity was inhibited in HAECs after vitamin E supplementation. Thus, we examined the effect of vitamin E on COX protein level. There are different isoenzymes of COX, a constitutive form (COX-1) and an inducible form (COX-2). Recently, a third form of COX, referred to as COX-3, was discovered in canine and human tissues, most notably the brain (19). The significance of this new member of the COX family requires further elucidation. As expected, COX-1 was similarly expressed regardless of stimulation, whereas COX-2 was barely expressed in unstimulated cells but greatly induced by IL-1ß (Fig. 5). However, vitamin E did not affect the expression of either form of COX, indicating that its effect on COX activity is post-translational.



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  		FIGURE 5 Effect of vitamin E on the expression of key enzymes involved in AA metabolism in HAECs. HAECs were preincubated with RRR-{alpha}-tocopherol for 20 h and then stimulated with IL-1ß (10 µg/L) for 18 h. COX-1, COX-2, cPLA2, and ß-actin (loading control) in cell lysates were determined in a Western blot assay. Results are representative of 3 independent experiments.

 
AA release is regulated by the action of PLA2, a family of enzymes serving as another rate-limiting factor in prostanoid biosynthesis. Although several forms of PLA2 exist, only cPLA2 is specific in catalyzing the hydrolysis of AA from membrane phospholipids. Therefore, we investigated whether the observed increase in AA release from HAECs was related to upregulation of cPLA2. cPLA2 was expressed in unstimulated cells and moderately enhanced by IL-1ß treatment (Fig. 5). Vitamin E dose dependently increased cPLA2 expression in both unstimulated and IL-ß–stimulated cells, with a more striking increase in the former, so that the levels of cPLA2 expression in unstimulated cells supplemented with higher levels of vitamin E matched those in the corresponding IL-1ß–stimulated cells (Fig. 5).

    Inhibition of cPLA2 prevents vitamin E–induced AA release. To further confirm our hypothesis that vitamin E increased AA release through upregulating cPLA2, we used a specific cPLA2 inhibitor, AACOCF3, to block cPLA2 activity before cells were supplemented with vitamin E. In this, as well as the following experiment, we used only unstimulated cells because, as demonstrated above, vitamin E had a similar effect on unstimulated and IL-1ß–stimulated cells. AACOCF3, in the absence of vitamin E, did not affect spontaneous AA release except at high concentration (5 µmol/L), where it slightly but significantly (P < 0.05) reduced AA release (Fig. 6). However, AACOCF3 inhibited (P < 0.05) the vitamin E–induced increase. The magnitude of inhibition did not differ among different doses of AACOCF3, indicating that the lowest dose (0.5 µmol/L) was adequate to completely block the increase in cPLA2 activity induced by vitamin E.



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  		FIGURE 6 Effect of inhibiting cPLA2 activity on vitamin E–induced AA release in HAECs. HAECs were incubated in the presence of RRR-{alpha}-tocopherol (40 µmol/L) and [3H]-AA (0.05 µCi/well) for 20 h and cPLA2 inhibitor AACOCF3 was added at different concentrations as indicated for another 5 h. AA release was calculated as described in Figure 4. Values are means ± SEM, n = 5. Means without a common letter (within lower- or uppercase) differ, P < 0.05. *Different from control, P < 0.05.

 
To ensure that the use of AACOCF3 did not confound the results by interfering with COX activity, we tested the effect of AACOCF3 at doses used in this study (0.5–5 µmol/L) on COX activity in the presence or absence of vitamin E (40 µmol/L). As shown before, vitamin E inhibited COX activity (P < 0.05); further, this inhibition was not affected by the presence of AACOCF3 at 0.5–5 µmol/L (Fig. 7A). This lack of AACOCF3 effect on COX activity was observed at different doses of vitamin E (Fig. 7B).



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  		FIGURE 7 Vitamin E–induced suppression in HAECs of COX activity in the presence of cPLA2 inhibitor. HAECs were incubated in the presence of RRR-{alpha}-tocopherol (40 µmol/L) for 20 h followed by addition of AACOCF3 at different concentrations as indicated for another 20 h (A). HAECs were incubated with a fixed concentration (0.5 µmol/L) of AACOCF3 and different concentrations of vitamin E as indicated (B). Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05.

 
To further confirm that the increase in prostanoid production was due to an increase in AA release, we assessed PGE2 production from endogenous AA in the presence or absence of the cPLA2 inhibitor, AACOCF3. When cPLA2 activity and thus AA release were inhibited, vitamin E no longer enhanced PGE2 production. On the contrary, it dose dependently inhibited PGE2 production (data not shown), an effect that was attributable to vitamin E–induced inhibition of COX activity.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The biosynthesis of prostanoids is accomplished in a metabolic cascade starting from its precursor fatty acid, AA. AA is present in membrane phospholipids and released under the hydrolytic action of PLA2. Released AA is metabolized to the unstable intermediate prostanoids by COX, also called prostaglandin H2 (PGH2) synthase. COX has bifunctional catalytic properties. It oxygenates and cyclizes AA to form prostaglandin G2 (PGG2) via its cyclooxygenase function, and then reduces PGG2 to PGH2 via its peroxidase function (20). The intermediate product PGH2 is then converted to different terminal prostanoids by the corresponding isomerases. Vitamin E interferes with AA metabolism at different stages, resulting in altered levels of prostanoid production, and this effect is tissue specific. For example, vitamin E increases PGI2 production in endothelial cells (21–24), but has no effect on pulmonary PGI2 production (25). Vitamin E inhibits TXA2 production in platelets (21,25), but has no effect on pulmonary TXA2 production (25). Vitamin E decreases PGE2 production in rat (26) and mouse (27,28) macrophages, and human mononuclear cells (29). However, Devaraj and Jialal (30,31) did not find an effect of vitamin E on PGE2 production in human monocytes.

Although some limited information is available regarding the effect of vitamin E on PGI2 production by endothelial cells, no information exists on the effect of vitamin E on the other vasodilator prostaglandin, PGE2. Furthermore, the underlying mechanisms have not been completely delineated. Previous work suggested that the effect might be mediated through PLA2, but the contribution of COX was not determined. In agreement with previous studies (21,22), the current study showed that vitamin E dose dependently (at 10–40 µmol/L) increased PGI2 production by HAECs. In addition, we observed a similar effect on PGE2 production, which implies a common mechanism governing the effect of vitamin E on both prostanoids.

Both PGI2 and PGE2 are products of the COX pathway in AA metabolism. Thus, we examined the effect of vitamin E on COX activity as the first step in determining the mechanism of the vitamin E–induced effect. However, the result did not explain the effect of vitamin E on PGI2 and PGE2 synthesis because vitamin E dose dependently inhibited COX activity despite its enhancement of PGI2 and PGE2 production. Because biosynthesis of prostanoids is determined not only by COX activity but also by the substrate availability, and vitamin E was reported to increase AA release and PLA2 activity in endothelial cells (22), we next examined the effect of vitamin E on AA release, which reflects mainly PLA2 activity. Our results confirmed a dose-dependent potentiating effect of vitamin E on AA release. Thus, vitamin E has an opposing effect on the 2 key regulatory enzymes in prostanoid biosynthesis, resulting in a net increase in production of vasodilator prostanoids in endothelial cells. These results also explain why the dose-dependent enhancement of PGI2 and PGE2 production by vitamin E was abated after vitamin E concentration was increased to a higher level of 60 µmol/L. IL-1ß–induced PGI2 production in the presence of vitamin E at 60 µmol/L was significantly lower than that at 20 or 40 µmol/L. A similar pattern was observed in IL-1ß–induced PGI2 production (Fig. 2). No further enhancement in AA release was observed after the concentration of vitamin E was increased to >40 µmol/L, whereas dose-dependent inhibition of COX activity continued above 40 µmol/L. Thus, increased AA availability plays a key role in determining vitamin E–induced upregulation of prostanoids up to 40 µmol/L. However, the promoting effect of vitamin E on AA release reaches its maximum at 40 µmol/L, whereas its inhibitory effect on COX activity continues at >40 µmol/L. As a result, as shown in Figure 2, vitamin E increased PGE2 and PGI2 production in a dose-dependent manner up to 40 µmol/L. After that point, no further increase is observed. In fact, prostanoid levels induced by vitamin E at 60 µmol/L are lower than those at 40 µmol/L. It would be interesting to determine whether a higher level of vitamin E would cause PGE2 and PGI2 production to drop below the control levels in culture. In support of this, when we used a cPLA2 inhibitor to block AA release, prostanoid production was dose dependently decreased by vitamin E. Because the plasma level of vitamin E at 40 µmol/L can be obtained in humans after a daily supplementation of 200 mg vitamin E, this result may suggest that this level of vitamin E provides the optimal level for increasing production of vasodilator prostonoids.

To delineate how vitamin E affects PLA2 and COX activity, we examined protein expression of each enzyme. In mammalian cells, there are at least 3 distinct families of PLA2 with different structures, subcellular distribution, and calcium requirements (32,33). However, the 85-kDa cPLA2 is believed to be mainly responsible for AA release because it selectively cleaves arachidonyl-containing phospholipids (34). We found that vitamin E dose dependently increased cPLA2 expression, and this effect was maximized at a dose of 40 µmol/L, which parallels its effect on AA release. Thus, it could be concluded that vitamin E enhances AA release by inducing cPLA2 expression. We further determined the effect of vitamin E on COX-1 (constitutive form) and COX-2 (inducible form) expression in HAECs. The results with HAECs were quite similar to those previously reported with macrophages (28,35,36), i.e., vitamin E inhibited COX activity but had no effect on COX-1 or COX-2 expression. We demonstrated that vitamin E inhibits COX activity in murine macrophages through reducing the production of peroxynitrite, an important cofactor for COX activity (36). Whether this mechanism also underlies the inhibition of COX activity in endothelial cells by vitamin E supplementation warrants investigation.

It is interesting to note that vitamin E inhibits PGE2 production in macrophages, as reported by us and others (26,28,36), but enhances PGE2 production in endothelial cells, as shown in the current study. This phenomenon is similar to that observed with nitric oxide (NO). Endothelium-derived NO, through the action of its endothelial NO synthase, is thought to maintain normal cardiovascular function, whereas macrophage-derived NO, through the action of inducible NO synthase, is considered a proinflammatory signal implicated in pathophysiological changes in the cardiovascular system (37). Vitamin E reduced NO production in splenocytes (38) and macrophages (36) but increased NO production in endothelial cells (39). The opposite effect of vitamin E on PGE2 production in endothelial cells and macrophages represents a desirable factor in endothelial homeostasis because endothelium-derived PGE2 helps maintain vasodilation, whereas macrophage-derived PGE2 is proinflammatory.

It is still not completely understood why vitamin E differentially affects PGE2 production in endothelial cells and macrophages. Sakamoto et al. (26) injected Wistar rats with vitamin E and found that both phorbol-12-myristate-13-acetate (PMA)- and A23187-induced PGE2 production in the macrophages isolated from these rats were prevented. They showed further that PMA or A23187 induced 14C-AA release in macrophages from control rats but did not do so in those from vitamin E–treated rats. However, they also reported that vitamin E did not affect LPS-induced PGE2 production under the same experimental condition (40). This stimulation-specific effect indicates that vitamin E may affect cPLA2 activity post-translationally, e.g., through its phosphorylation. The observation that vitamin E blocks PMA-induced AA release may be due to its inhibition of protein kinase C (PKC) activation. Devaraj and Jialal (31) showed that vitamin E inhibited PKC activity in human monocytes. In contrast, in the current study, vitamin E enhanced cPLA2 activity at the translational level because a dose-dependent increase in expression of this protein (Fig. 5) occurred. Further studies are warranted to investigate the mechanism by which vitamin E increases cPLA2 synthesis.

In conclusion, vitamin E had an opposite effect on 2 rate-limiting steps in the biosynthesis of the vasodilator prostanoids PGI2 and PGE2 in HAECs, i.e., increasing substrate AA release and inhibiting COX activity. The net effect was an increased production of both prostanoids, indicating that substrate availability is the predominant factor through which vitamin E increases prostanoid production under these experimental conditions. Further studies are required to determine the net effect of higher levels of vitamin E on prostanoid synthesis. The vitamin E–induced AA release is due to increased cPLA2 activity, which, in turn, is due to a higher level of cPLA2 expression. In contrast, the inhibitory effect of vitamin E on COX activity is not mediated through inhibition of COX-1 or COX-2 expression. The vitamin E–induced increase in PGI2 and PGE2 production may contribute to its suggested beneficial effect in preserving endothelial function.


   ACKNOWLEDGMENTS
 
The authors thank Stephanie Marco for the preparation of the manuscript.


   FOOTNOTES
 
1 Presented in part at Experimental Biology 04, April 17–21, 2004, Washington, DC [Wu, D., Liu, L., Meydani, M. & Meydani, S. N. (2004) Vitamin E (E) increases prostacyclin (PGI2) and prostaglandin (PG)E2 production by human aorta endothelial cell (HAEC) by differentially modulating cyclooxygenase (COX) and phospholipase A2 (PLA2) activities. FASEB J. 18: A860 (abs.)]. Back

2 Supported by the U.S. Department of Agriculture, Agriculture Research Service, under contract number 53-K06-01. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. Back

4 Abbreviations used: AA, arachidonic acid; AACOCF3; arachidonyltrifluoromethyl ketone; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; CVD, cardiovascular disease; FBS, fetal bovine serum; HAEC, human aortic endothelial cell; IL-1, interleukin-1; PGE2, prostaglandin E2; PGG2, prostaglandin G2; PGH2, prostaglandin H2; PGI2, prostaglandin I2, prostacyclin; PKC, protein kinase C; PLA2, phospholipase A2; PMA, phorbol-12-myristate-13-acetate; TXA2, thromboxane A2. Back

Manuscript received 20 April 2005. Initial review completed 6 May 2005. Revision accepted 18 May 2005.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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