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Supplement

The Effect of -Tocopherol on Monocyte Proatherogenic Activity¹

Ishwarlal Jialal^*,2, Sridevi Devaraj^* and Nalini Kaul^*

Departments of ^* Pathology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9073

²To whom correspondence should be addressed. E-mail: jialal.i{at}pathology.swmed.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
Atherosclerosis is the leading cause of morbidity and mortality in Westernized populations. The monocyte is a crucial cell in the genesis of the atherosclerotic lesion and is present during all stages of atherosclerosis. -Tocopherol (AT) is the most active component of the vitamin E family and is the principal and most potent lipid-soluble antioxidant in plasma and LDL. With regard to monocyte function, AT supplementation (1200 IU/d) has been shown to decrease release of reactive oxygen species, lipid oxidation, release of cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-) and decrease adhesion of monocytes to human endothelium. The mechanism of inhibition of superoxide and lipid oxidation by monocytes appears to be via inhibition of protein kinase C (PKC), the decrease in IL-1ß and TNF- release by inhibition of 5-lipoxygenase and the inhibition of monocyte-endothelial cell adhesion via decrease in adhesion molecules on monocytes, CD11b and VLA-4 and by decreasing DNA-binding activity of nuclear transcription factor B. Thus, in addition to the decrease in oxidative stress resulting from AT supplementation, as evidenced by decreased F₂-isoprostanes and LDL oxidizability, AT is anti-inflammatory and exerts beneficial antiatherogenic effects on cells crucial in atherogenesis such as monocytes.

KEY WORDS: • -tocopherol • monocytes • vitamin E • inflammation • antioxidant

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
Atherosclerosis is the leading cause of morbidity and mortality in Westernized populations. Atherosclerotic lesions are classified as fatty streaks, fibrous plaques and complex lesions (Berliner et al. 1995 , Ross 1999 ). The ubiquitous fatty streak lesion is the earliest lesion in atherosclerosis; it is a grossly flat, lipid-rich lesion consisting mainly of monocyte-macrophages and some smooth muscle cells. The fatty streak may regress or progress into the fibrous plaque, which is the characteristic lesion of atherosclerosis and comprised mainly of intimal smooth muscle cells, macrophages and T-lymphocytes, surrounded by connective tissue matrix and containing variable amounts of intracellular and extracellular lipid. At the lumen of the artery, this lesion is generally covered by a fibrous cap of smooth muscle and connective tissue. Beneath the fibrous cap, the lesions are highly cellular and contain a necrotic core of smooth muscle, macrophages, which contain lipid droplets, connective tissue and T lymphocytes. Fibrous plaques may undergo calcification, fissuring and/or thrombosis to form the complex lesion that is associated with clinical atherosclerosis. Thus, it appears that the major cellular participants in the atherosclerotic process include an active vascular endothelium, smooth muscle cells, blood-borne cells such as monocyte-macrophages, T lymphocytes and platelets. This review will focus only on the proatherogenic properties of monocytes and their modulation with the lipid-soluble antioxidant, -tocopherol.

After endothelial dysfunction induced by factors including LDL, homocysteine, hypertension or diabetes, monocytes attach to the endothelium via the interaction of adhesion molecules and counterreceptors on the endothelium and monocytes. Monocytes are then attracted into the subendothelial space where they take up lipid and become foam cells, the hallmark of the fatty streak lesion(Ross 1999 ).

	Oxidized LDL hypothesis

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
The monocyte is therefore a crucial cell in the genesis of the atherosclerotic lesion and is present during all stages of atherosclerosis. The importance of monocyte-macrophages in atherogenesis is further underscored by the preliminary findings of certain altered monocyte functions in hypercholesterolemia, a known risk factor for coronary artery disease. Hypercholesterolemia has been shown to be associated with an abnormally high number of morphologically abnormal monocyte/macrophages in the bloodstream. LDL, which may be modified by oxidation, glycation, aggregation, association with proteoglycans or incorporation into immune complexes, is a major cause of injury to endothelium. One of the major and most plausible modifications of LDL is oxidation (Devaraj and Jialal 1996 , Steinberg et al. 1989 , Witztum and Steinberg 1991 ). Several lines of evidence support a proatherogenic role for oxidized LDL (Ox-LDL).³ All major cells of the artery wall such as monocyte-macrophages, endothelial cells and smooth muscle cells can modify LDL oxidatively in vitro. Ox-LDL is not recognized by the LDL receptor but is taken up avidly by the scavenger receptor on the macrophages. The free radical peroxidation of LDL lipids results in numerous structural changes, all depending on a common initiating event, i.e., the peroxidation of polyunsaturated fatty acids (PUFA) in LDL. Some of the properties of Ox-LDL include increased negative charge, increased density, enrichment in lipid peroxides, loss of esterified cholesterol, fragmentation of apolipoprotein (apo) B and decreased PUFA content. The biologic effects reported to date for Ox-LDL contribute to the atherogenic process. Ox-LDL is cytotoxic and can result in endothelial dysfunction and the promotion of the fatty streak into a more complex lesion. Ox-LDL is a potent chemoattractant for circulating monocytes. In the initial phase of oxidation, minimally modified LDL (MM-LDL) is formed in the subendothelial space. MM-LDL has been shown to stimulate binding of monocytes to endothelial cells. Also, MM-LDL stimulates production of monocyte chemotactic protein-1 (MCP-1), which promotes monocyte chemotaxis. These molecular events result in monocyte binding to the endothelium and its subsequent migration into the subendothelial space where MM-LDL stimulates production of colony stimulating factors, such as monocyte colony stimulating factor (M-CSF). M-CSF promotes the differentiation of monocytes into macrophages. The macrophages can then further oxidize MM-LDL to Ox-LDL. Ox-LDL is also an inhibitor of monocyte migration and thereby promotes retention of monocyte macrophages in the artery wall. In addition, LDL oxidizability has been shown to be increased with other established coronary artery disease (CAD) risk factors, such as diabetes, smoking, hypertension and hyperlipidemia. Several lines of evidence support the in vivo existence of Ox-LDL (Devaraj and Jialal 1996 , Steinberg et al. 1989 , Witztum and Steinberg 1991 ). Antibodies to Ox-LDL recognize material from atherosclerotic lesions, but not from normal arteries. Circulating autoantibodies to Ox-LDL have been demonstrated to be increased in diabetics, patients with renal failure and hypertensive subjects, with a concomitant increase in LDL oxidation. The presence of autoantibodies to Ox-LDL has been correlated positively with the progression of atherosclerosis, as manifested by carotid artery stenosis (Salonen et al. 1992 ). It has also been shown that the susceptibility of LDL to oxidation, i.e., the lag phase, varied proportionately with the severity of atherosclerosis as evaluated by angiography (Regnstrom et al. 1992 ). However, the most persuasive data comes from animal models of atherosclerosis in which studies with antioxidants such as probucol, -tocopherol (AT), BHT and N,N'-diphenyl phenylene diamine (DPPD) show a significant decrease in the degree of LDL oxidation and the extent of atherosclerotic lesions. However, the toxicity of BHT and DPPD limits their use in humans. In addition, probucol use in humans has been shown to lower HDL cholesterol and prolong the Q-T interval, which may cloud its potential for long-term use in humans.

	Apo-E–deficient mice and atherosclerosis: role of monocyte-macrophages

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
Recent exciting evidence for the pivotal role of monocyte-macrophages in atherogenesis comes from studies in apo E knockout mice. Apo E knockout mice are hypercholesterolemic; unlike other rodent models of atherosclerosis, they develop atherosclerosis spontaneously with consumption of a low fat diet (Smith et al. 1995 ). The hypercholesterolemia and atherosclerosis in these mice are diet responsive, in that they are increased by consumption of a high fat, Western-type diet. Remarkably, these lesions are representative of human atherosclerosis in that they progress with age from early fatty streaks to complex fibrous plaques with necrotic cores and are found at the same sites of predilection as human lesions. Similar to human and rabbit lesions, oxidized lipoprotein epitopes are found in lesions of apo E–deficient mice, and sera from these mice contain high titers of autoantibodies that recognize oxidized lipoproteins and can stain lesions in rabbits (O’Neill 1997 ). The apo E–deficient mice are therefore an attractive model system in which to elicit factors that modulate the pathogenesis of atherosclerosis. In these mice, it has been shown that additional knock out of the gene for M-CSF, despite severe hypercholesterolemia, results in decreased levels of monocytes in their peripheral blood, reduced tissue macrophages and significantly less atherosclerosis in the proximal aorta (Qiao et al. 1997 , Smith et al. 1995 ). Furthermore, additional knockout of CCR2 (the receptor for monocyte chemotactic protein-1) in the apo E–deficient mice significantly decreases lesion formation (Boring et al. 1998 ). Further evidence for the role of monocyte-macrophages in atherosclerosis comes from studies in LDL receptor–deficient mice in which additional knockout of MCP-1 resulted in less lipid deposition in aortas, fewer macrophages in the arterial wall and reduced atherosclerosis (Osuga et al. 1998 ).

	-Tocopherol

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
Vitamin E exists as at least eight naturally occurring compounds, including -, ß-, - and -tocopherol and -, ß-, - and -tocotrienol (Traber and Sies 1996 ). AT is the most active component of the vitamin E family and occurs naturally as one isomer; it is the principal and most potent lipid-soluble antioxidant in plasma and LDL. Several studies show that low levels of AT are associated with increased atherosclerosis and that increased intakes of AT appear to be protective against CAD (Diaz et al. 1997 , Jialal and Grundy 1992 ). Epidemiological studies strongly favor AT supplementation; however, results from prospective studies, though promising, are equivocal (Pryor, 2000 ). Steiner et al. (1995) showed that patients who received AT and aspirin had a significant reduction in transient ischemic attacks and ischemic strokes compared with those taking aspirin alone. The ATBC study and HOPE studies did not show a distinct benefit for AT supplementation; however, as reviewed previously, the former used a low dose and the latter was conducted in several countries (Jialal and Devaraj 2000 , Pryor, 2000 ). The CHAOS and GISSI studies (four-way analyses) showed that AT supplementation was associated with significant reduction in nonfatal myocardial infarction and cardiovascular deaths, respectively (Jialal et al. 2000 , Pryor 2000 ). Thus, the totality of evidence appears to indicate the benefits of AT supplementation, and larger clinical trials with defined end points and doses will determine the utility of AT supplementation in the reduction of CAD.

	-Tocopherol and oxidation

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
AT has been shown to inhibit LDL oxidation in vitro and in vivo (Devaraj and Jialal 1996 , Jialal and Grundy 1992 ). AT supplementation has been found to decrease F₂-isoprostanes and atherogenesis in apo E–deficient mice (Pratico et al. 1998 ). In human subjects, AT supplementation has been shown to lower urinary F₂-isoprostanes in subjects with hypercholesterolemia and in diabetic individuals (Davi et al. 1997 and 1999 ). Also, in a recent report, we showed that AT supplementation (400 IU/d) can decrease urinary F₂-isoprostane levels (Marangon et al. 1999 ).

	-Tocopherol and monocyte function

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
-Tocopherol has also been shown to exert potential antiatherogenic effects on crucial cells in atherosclerosis such as endothelial cells, smooth muscle cells, platelets and monocytes (Devaraj and Jialal 1998 ). In addition to decreasing LDL oxidizability, it has been shown to decrease release of reactive oxygen species (ROS) and proinflammatory cytokines and to inhibit monocyte-endothelial cell adhesion.

Reactive oxygen species.

Monocytes, when appropriately stimulated, can produce many biologically active mediators that can influence virtually all aspects of atherogenesis. Monocytes have been shown to induce peroxidation of lipids such as in LDL, by the generation of ROS, including superoxide anion, hydrogen peroxide, hydroxyl radical, peroxynitrite, myeloperoxidase and hypochlorous acid. Cathcart et al. (1985 and 1989) showed that activated human monocytes can oxidize LDL in vitro and that superoxide anion participated in both the oxidation of LDL and its conversion to a cytotoxin because addition of superoxide dismutase (SOD) significantly inhibited monocyte-mediated LDL oxidation. They also showed that in vitro incubation of monocytes with BHT, vitamin E or glutathione significantly reduced oxidation of LDL and formation of cytotoxic LDL (Cathcart et al. 1985 ). In recent years, considerable evidence has accumulated supporting a role for ROS as activators of signaling pathways, such as the mitogen-activated protein kinase pathways, resulting in phosphorylation and activation of transcription factors, such as nuclear transcription factor B (NFB) and AP-1. Activated transcription factors then alter gene expression, resulting in new cellular outcomes, including apoptosis and cell growth. There is a paucity of data examining the role of AT supplementation on ROS release from human monocytes. We showed that supplementation with AT (1200 IU/d) for 3 mo resulted in a twofold enrichment of monocytes with AT and significantly reduced the release of superoxide anion (59%) as well as hydrogen peroxide (50%) in resting and lipopolysaccharide (LPS)-activated monocytes (Devaraj and Jialal 1996 ). Because we were not able to isolate LDL from the same patients, we examined the ability of monocytes to oxidize lipid by utilizing an artificial lipoprotein emulsion. This emulsion contained cholesterol linoleate, cholesterol arahidonate and cholesterol oleate/bovine serum albumin in the proportion that would normally be present in human LDL. This emulsion has previously been shown to be taken up avidly by macrophages, resulting in foam cell formation and ceroid accumulation. In the presence of the artificial lipoprotein emulsion, there was a 1.5-fold increase in lipid oxidation by activated cells. After AT supplementation, there was a 40% decrease in lipid oxidation by the activated monocytes. Thus, this study showed that AT supplementation, in addition to partitioning in LDL and decreasing oxidative susceptibility, also results in enrichment of monocytes with subsequent decrease in lipid oxidation.

We showed recently that LPS-activated monocytes from type 2 diabetic subjects with and without macrovascular disease released increased levels of superoxide anion compared with matched controls (Devaraj and Jialal 2000 ). AT supplementation (1200 IU/d) significantly decreased superoxide anion release in control as well as diabetic monocytes to the same degree. Recently, van Tits et al. (2000) showed that supplementation with RRR-AT (600 IU/d) significantly inhibited superoxide production by polymorphonuclear leukocytes (PMN) activated with phorbol ester, but not oxidized LDL or opsonized zymosan. Mechanisms for inhibition, however, were not explored.

One common mechanism to account for the effects of AT may be through inhibition of protein kinase C (PKC) (Azzi et al. 1998 ). PKC plays a major role in signal transduction; it is implicated in a variety of events ranging from respiratory burst, to platelet aggregation and cellular differentiation. Many oxidant-initiated signaling processes are known to involve PKC. Because Li and Cathcart (1994) had shown previously that PKC mediated superoxide anion release and lipid oxidation by monocytes, we examined the effects of specific PKC inhibitors on these parameters, to gain some insights on the effect of AT supplementation. We used two specific inhibitors, Calphostin C, a regulatory subunit inhibitor of PKC, and Bis-indoleyl maleimide, which binds to the catalytic subunit and inhibits PKC activity. Also, at the concentrations used in our experiments, these specific inhibitors did not show any antioxidant properties or cytotoxicity. Calphostin C as well as Bis-indoleyl maleimide produced an average 50% decrease in superoxide anion release and a 32% decrease in lipid oxidation by activated human monocytes, which could explain in large part the inhibition seen with AT supplementation. Hence, it appears that the inhibition in superoxide anion release and lipid oxidation by activated human monocytes after AT supplementation could be attributed to an inhibition of PKC activity rather than a general antioxidant effect (Devaraj and Jialal 1996 and 2000 ). Furthermore, Li and Cathcart (1999) recently showed that superoxide generation and lipid oxidation in activated human monocytes appears to be regulated by PKC-. In addition, results from an in vitro study by Cachia et al. (1998) reveal that AT inhibits superoxide production by monocytes by impairing the assembly of NADPH oxidase, the enzyme responsible for generating the respiratory burst. AT inhibits p47phox translocation to the membrane and also impairs phosphorylation of p47phox. This study also suggests that inhibition of PKC activity is not directly due to the antioxidant capacity of AT, but requires AT integration into the cell membrane where it can interact directly with PKC and NADPH oxidase.

Cytokine release.

Macrophages secrete several proinflammatory, proatherogenic cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)- (Clinton and Libby 1992 , Libby and Hanson 1991 , Raines and Ross 1996 ). IL-1ß has been shown to be present in the atherosclerotic lesion and is found to be increased in patients with ischemic cardiomyopathy. IL-1ß has been shown to augment monocyte-endothelial cell adhesion, increase procoagulant activity, promote cholesterol esterification in macrophages and stimulate smooth muscle cell proliferation (Devaraj and Jialal 1999b ). TNF- is a multifunctional cytokine that exerts pleiotropic biological actions. It activates endothelial cells, stimulates angiogenesis and induces smooth muscle cell proliferation. Increased mRNA for TNF- has been documented in aortae of Watanabe heritable hyperlipidemic rabbits and carotid atherosclerotic plaques. TNF- processing via its receptor can promote apoptosis and thus could contribute to the necrotic core of the atherosclerotic lesion. Akeson et al. (1991) showed in vitro that incubation of THP-1 cells with AT significantly inhibits phorbol myristate acetate (PMA)-induced IL-1ß secretion. With regard to cytokine release, we showed that AT supplementation (1200 IU/d for 3 mo) resulted in a 90% inhibition of IL-1ß release from LPS-activated monocytes (Devaraj and Jialal 1996 ). Cannon et al. (1991) showed previously that AT supplementation (800 IU/d) prevented the increase in IL-1ß release from LPS-activated mononuclear cells after exercise; however, they did not elucidate the mechanism of inhibition. Subsequently, we explored different mechanisms by which AT inhibited IL-1ß release (Devaraj and Jialal 1999 ).The potential mechanisms that were examined included its effect as a general antioxidant, its inhibitory effect on PKC activity and its effects on the cyclooxygenase (COX)-lipoxygenase pathways. Because AT in a chain-breaking antioxidant that provides membrane integrity, it could prevent induction of IL-1ß release by decreasing ROS. Using encapsulated SOD as well as catalase, we failed to inhibit IL-1ß release from human monocytes despite a substantial increase in cellular SOD activity. Also, ß-tocopherol, a similar antioxidant, had no effect on IL-1ß release from human monocytes. Thus, AT did not appear to act through its antioxidant mechanism in inhibiting IL-1ß release. Furthermore, although AT decreased PKC activity in human monocytes, the inhibition of PKC as elucidated with the two specific inhibitors, Calphostin C and Bis-indoleylmaleimide, did not result in any change in IL-1ß release (Devaraj and Jialal 1996 and 1999b ). Leukotriene (LT) B₄, the main product of the 5-lipoxygenase (5-LO) pathway, has been shown to induce IL-1ß secretion from human monocytes (Rola-Plesczynski and Lemaire 1985 ). Also, AT has been shown to inhibit 5-LO activity in cell-free systems and in rat neutrophils (Chan 1989 , Goetzl 1990, Reddanna et al. 1985 and 1989 ). AT has been shown to inhibit 5-LO purified from potato tubers, and this effect was unrelated to its antioxidant function (Reddanna et al. 1985 ). In addition, RRR-AT supplementation (800 IU/d) significantly decreased 5-LO in normal volunteers as evidenced by a reduction in urinary LTE₄, a product of 5-LO (Denzlinger et al. 1995 ). Hence, we tested the effect of AT on LTB₄ levels in activated human monocytes. AT significantly inhibited LTB₄ and IL-1ß release from activated human monocytes, and this was reversed with LTB₄. We also tested the effect of specific inhibitors of 5-LO, such as MK886 (binds 5-LO activating protein and blocks membrane translocation of 5-LO and its subsequent activation) and REV-5901 (peptido-leukotriene receptor antagonist, inhibits 5-LO due to its structural similarity) on IL-1ß release from activated human monocytes. Both inhibitors significantly decreased IL-1ß release to the same extent as AT. Thus, AT inhibited IL-1ß release from human monocytes via inhibition of 5-LO. We also examined the effect of AT on prostaglandin (PG) E₂, a COX-derived metabolite of arachidonic acid. There was no effect of AT on PGE₂ release, and indomethacin, a COX inhibitor, had no effect on IL-1ß release from activated human monocytes. To examine whether AT had an effect on IL-1ß synthesis, both mRNA expression as well as stability were measured in the presence of AT. There was no change in expression or stability of mRNA for IL-1ß in the presence of AT. Thus, we elucidated a novel biological effect of AT, i.e., inhibition of IL-1ß from activated human monocytes via inhibition of the 5-LO pathway post-transcriptionally (Devaraj et al. 1999 ). Recently, we have also shown by Western blotting with specific antibodies to 5-LO that AT significantly inhibits translocation of 5-LO from the cytosol to the membrane.

In the same supplementation study in healthy volunteers (Devaraj and Jialal 1996 ) as well as in a subsequent study in diabetic subjects (Devaraj and Jialal 2000 ), we showed that AT supplementation also resulted in a significant inhibition in IL-1ß and TNF- release from LPS-activated human monocytes. Studies are being conducted to elucidate the mechanism of TNF- inhibition by AT. Support for our data comes from the work of van Tits et al. (2000) who showed recently that supplementation with RRR-AT (600 IU/d) significantly inhibited the release of IL-1ß and TNF- from activated mononuclear cells.

Monocyte-endothelial cell adhesion.

An early event in the genesis of the fatty streak lesion is the attachment of monocytes to vascular endothelium (Adams and Shaw 1994 , Ross 1999 ). After adhesion of monocytes to endothelium, they migrate into the intima, imbibe lipid and become foam cells. The exact mechanism by which monocyte-endothelial cell adhesion occurs in vivo remains to be elucidated. Recent work has identified the specific adhesion molecules on endothelial cells and monocytes. These adhesion molecules include E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells and members of the ß-2 integrin family, CD11a, CD11b, CD11c/18 and the ß-1 integrin, VLA-4(CD49d/29) on monocytes. The role of cell adhesion molecules (CAM) in leukocyte-endothelial interactions and regulation of leukocyte migration has been reviewed extensively by Adams and Shaw (1994) . On leukocytes, the CD11a and CD11b integrins as well as L-selectin have been implicated. In human atheroma, VCAM-1 and ICAM-1 are expressed on endothelial cells and to a smaller extent, on smooth muscle cells and macrophages.

Modulation of monocyte-endothelial adhesion could be an important target in atherosclerosis. It has also been shown that agonist-induced adhesion of monocytes to endothelium is mediated by translocation of the transcription factor, NFB (Brand et al. 1996 , Collins 1993 ). Recent findings indicate the involvement of ROS in the pathway of NFB activation (Schreck et al. 1991 ). NFB is a mammalian transcription factor that is directly involved in the activation of genes responsible for immune, inflammatory or acute phase responses. In cells that have inducible NFB activity, the factor comprises a p50-p65 heterodimer bound by an inhibitory subunit called IB in the cytosol, and can be activated by dissociating IB. After induction by stimuli such as PMA, LPS, IL-1or TNF, the activated NFB translocates to the nucleus where it binds to the B-specific DNA sequence. The importance of activated NFB in atherosclerosis has been demonstrated by its presence in smooth muscle cells, macrophages and endothelial cells of human atherosclerotic lesion tissue, but not in normal vessels (Brand et al. 1996 ). A variety of genes are induced in the atherosclerotic lesion that have been shown to be regulated by NFB, including genes encoding TNF, IL-1, tissue factor, M-CSF, VCAM-1 and ICAM-1. Baeuerle (1991) suggested a novel signal transduction pathway for NFB activation involving ROS as second messengers on the basis of studies with Jurkat T cells that responded to the addition of exogenous hydrogen peroxide. Also, MM-LDL has also been shown to activate NFB in endothelial cells. Some of the antioxidants that have been shown to inhibit NFB activation include pyrollidone dithiocarbamate, N-acetyl cysteine and -lipoate. Suzuki and Packer (1993) showed that NFB activation can be inhibited in TNF-activated Jurkat T cells by treatment with AT acetate and succinate, resulting in decreased DNA binding activity. However, they did not observe any inhibition with AT. Although studies have shown that AT enrichment of endothelial cells inhibits subsequent monocyte-endothelial cell adhesion, we have shown that AT supplementation (1200 IU/d for 3 mo) resulted in a significant inhibition in monocyte-endothelial cell adhesion (Devaraj and Jialal 1996 ). Furthermore, we have shown that AT enrichment of U937 monocytic cells with AT (50 or 100 µmol/L) significantly decreases monocyte-endothelial cell adhesion by inhibiting the expression of counter-receptors CD11b A and VLA-4 on the monocytes as well as by inhibiting the activation of the transcription factor, NFB (Islam et al. 1998 ). Furthermore, we show that SN50, a cell-permeable inhibitor of NFB translocation, significantly decreases monocyte-endothelial cell adhesion, thereby confirming the role of NFB in monocyte-endothelial cell adhesion. Martin et al. (1997) showed that in vitro enrichment of human aortic endothelial cells with AT significantly inhibited LDL-induced adhesion of monocytes to endothelial cells in a dose-dependent manner with a concomitant reduction in levels of sICAM. Furthermore, we showed that SN50, a cell-permeable inhibitor of NFB translocation, significantly decreases monocyte-endothelial cell adhesion, thereby confirming the role of NFB in monocyte-endothelial cell adhesion. Subsequently, Yoshida et al. (1999) showed that AT supplementation at lower doses (600 mg/d for 10 d) significantly inhibited surface expression of CD11b on activated PMN as well as adhesion of activated PMN to human umbilical vein endothelial cells (HUVEC), and this was correlated inversely with serum AT concentrations. Unlike our findings with monocytes, they appear to implicate inhibition of PKC by AT in human PMN.

Increasing evidence supports the role of plasma levels of CAM, sICAM-1, sVCAM-1 and E-selectin as molecular markers of atherosclerosis. Increased levels of CAM have been observed in atherosclerotic plaques. Several epidemiologic studies have shown a strong association between levels of soluble CAM and coronary as well as carotid atherosclerosis. (Devaraj and Jialal 1999a , Hwang et al. 1997 , Ridker et al. 1998 , Rohde et al. 1998 ) With regard to modulation of CAM levels with antioxidants, the data are sparse. Pretreatment of HUVEC or LDL with AT significantly reduced expression of VCAM-1 on HUVEC induced by oxidized LDL in vitro (Cominacini et al. 1997 ). Recently, it was shown that supplementation with N-acetyl-L-cysteine resulted in an increase in erythrocyte glutathione and a concomitant reduction in plasma VCAM-1 levels (DeMattia et al. 1998 ). However, there is a paucity of data on the effect of AT supplementation on soluble CAM levels. We have recently shown that monocytes from type 2 diabetic subjects with and without macrovascular disease are more proatherogenic than are those of matched controls as assessed by increased levels of superoxide, IL-1ß and greater adhesion to endothelium (Devaraj and Jialal 2000 ). AT supplementation (1200 IU/d) significantly reduced monocyte-pro-atherogenic activity. In addition, AT supplementation also resulted in a significant decrease in levels of soluble adhesion molecules, ICAM, VCAM and E-selectin (Devaraj and Jialal 2000 ).

Other cardiovascular benefits of -tocopherol.

AT may possess other beneficial effects on critical cells in atherogenesis (Devaraj and Jialal 1998 ). AT supplementation has been shown to decrease platelet aggregation and improve endothelium-dependent vasodilation.

	Summary

TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 
Thus, with regard to monocyte function, AT supplementation (1200 IU/day) has been shown to decrease the release of ROS, lipid oxidation, release of cytokines such as IL-1ß and TNF- and decrease adhesion of monocytes to human endothelium. The mechanism of inhibition of superoxide and lipid oxidation by monocytes by AT appears to be via inhibition of PKC, the decrease in IL-1ß and TNF- release by inhibition of 5-LO and the inhibition of monocyte-endothelial cell adhesion via decrease in adhesion molecules, CD11b and VLA-4 on monocytes, via inhibition of NFB. In addition to the decrease in oxidative stress resulting from AT supplementation as evidenced by decreased F₂-isoprostanes and LDL oxidizability, its anti-inflammatory effects on monocytes could further explain its antiatherogenic effects.

	ACKNOWLEDGMENTS

 
Studies cited in this review were supported by NIH grants R01 AT00005 and K24 AT00596.

	FOOTNOTES

 
¹ Presented as part of the symposium, Molecular Mechanisms of Protective Effects of Vitamin E in Atherosclerosis, given at Experimental Biology 2000, April 16, 2000 in San Diego, CA. This symposium was sponsored by the American Society for Nutritional Sciences and was supported by an educational grant from Archer Daniels Midland Company and BASF corporation. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Mohsen Meydani, Tufts School of Nutrition Science and Policy and Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA and Maret G. Traber, Linus Pauling Institute, Oregon State University, Corvallis, OR and University of California, Davis, School of Medicine, Sacramento, CA.

³ Abbreviations used: apo, apolipoprotein; AT, -tocopherol; CAD, coronary artery disease; CAM, cell adhesion molecules; COX, cyclooxygenase; DPPD, N,N'-diphenyl phenylene diamine; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; 5-LO, 5-lipoxygenase; LPS, lipopolysaccharide; LT, leukotriene; M-CSF, monocyte colony stimulating factor; MCP-1, monocyte chemotactic protein-1; MM-LDL, minimally modified LDL; Ox-LDL, oxidized LDL; NFB, nuclear transcription factor B; PG, prostaglandin; PKC, protein kinase C; PMN, polymorphonuclear leukocyte; PUFA, polyunsaturated fatty acids; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1.

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TOP ABSTRACT INTRODUCTION Oxidized LDL hypothesis Apo-E-deficient mice and... {alpha}-Tocopherol {alpha}-Tocopherol and oxidation {alpha}-Tocopherol and monocyte... Summary REFERENCES

 

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