Angelo Azzi

Vitamin E: non-antioxidant roles.

Vitamin E was originally considered a dietary factor of animal nutrition especially important for normal reproduction. The significance of vitamin E has been subsequently proven as a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life processes. More recently alpha-tocopherol has been found to possess functions that are independent of its antioxidant/radical scavenging ability. Absorption in the body is alpha-tocopherol selective and other tocopherols are not absorbed or are absorbed to a lesser extent. Furthermore, pro-oxidant effects have been attributed to tocopherols as well as an anti-nitrating action. Non-antioxidant and non-pro-oxidant molecular mechanisms of tocopherols have been also described that are produced by alpha-tocopherol and not by beta-tocopherol. alpha-Tocopherol specific inhibitory effects have been seen on protein kinase C, on the growth of certain cells and on the transcription of some genes (CD36, and collagenase). Activation events have been seen on the protein phosphatase PP2A and on the expression of other genes (alpha-tropomyosin and Connective Tissue Growth Factor). Non-antioxidant molecular mechanisms have been also described for gamma-tocopherol, delta-tocopherol and tocotrienols.

Non-Antioxidant Activities of Vitamin E

Molecules in biological systems often can perform more than one function. In particular, many molecules have the ability to chemically scavenge free radicals and thus act in the test tube as antioxidant, but their main biological function is by acting as hormones, ligands for transcription factors, modulators of enzymatic activities or as structural components. In fact, oxidation of these molecules may impair their biological function, and cellular defense systems exist which protect these molecules from oxidation. Vitamin E is present in plants in 8 different forms with more or less equal antioxidant potential (alpha-, beta-, gamma-, delta-tocopherol/tocotrienols); nevertheless, in higher organisms only alpha-tocopherol is preferentially retained suggesting a specific mechanism for the uptake for this analogue. In the last 20 years, the route of tocopherol from the diet into the body has been clarified and the proteins involved in the uptake and selective retention of alpha-tocopherol discovered. Precise cellular functions of alpha-tocopherol that are independent of its antioxidant/radical scavenging ability have been characterized in recent years. At the posttranslational level, alpha-tocopherol inhibits protein kinase C, 5-lipoxygenase and phospholipase A2 and activates protein phosphatase 2A and diacylglycerol kinase. Some genes (e. g. scavenger receptors, alpha-TTP, alpha-tropomyosin, matrix metalloproteinase-19 and collagenase) are modulated by alpha-tocopherol at the transcriptional level. alpha-Tocopherol also inhibits cell proliferation, platelet aggregation and monocyte adhesion. These effects are unrelated to the antioxidant activity of vitamin E, and possibly reflect specific interactions of alpha-tocopherol with enzymes, structural proteins, lipids and transcription factors. Recently, several novel tocopherol binding proteins have been cloned, that may mediate the non-antioxidant signaling and cellular functions of vitamin E and its correct intracellular distribution. In the present review, it is suggested that the non-antioxidant activities of tocopherols represent the main biological reason for the selective retention of alpha-tocopherol in the body, or vice versa, for the metabolic conversion and consequent elimination of the other tocopherols.

Non-antioxidant molecular functions of α-tocopherol (vitamin E)

α‐Tocopherol (the major vitamin E component) regulates key cellular events by mechanisms unrelated with its antioxidant function. Inhibition of protein kinase C (PKC) activity and vascular smooth muscle cell growth by α‐tocopherol was first described by our group. Later, α‐tocopherol was shown to inhibit PKC in various cell types with consequent inhibition of aggregation in platelets, of nitric oxide production in endothelial cells and of superoxide production in neutrophils and macrophages. α‐Tocopherol diminishes adhesion molecule, collagenase and scavenger receptor (SR‐A and CD36) expression and increases connective tissue growth factor expression.

Vitamin E Reduces the Uptake of Oxidized LDL by Inhibiting CD36 Scavenger Receptor Expression in Cultured Aortic Smooth Muscle Cells

BACKGROUND Vitamin E is well known as an antioxidant, and numerous studies suggest that it has a preventive role in atherosclerosis, although the mechanism of action still remains unclear. METHODS AND RESULTS The original aim of this study was to establish whether alpha-tocopherol (the most active form of vitamin E) acts at the earliest events on the cascade of atherosclerosis progression, that of oxidized LDL (oxLDL) uptake and foam-cell formation. We show here that the CD36 scavenger receptor (a specific receptor for oxLDL) is expressed in cultured human aortic smooth muscle cells (SMCs). Treatment of SMCs and HL-60 macrophages with alpha-tocopherol (50 micromol/L, a physiological concentration) downregulates CD36 expression by reducing its promoter activity. Furthermore, we find that alpha-tocopherol treatment of SMCs leads to a reduction of oxLDL uptake. CONCLUSIONS This study indicates that CD36 is expressed in cultured human SMCs. In these cells, CD36 transports oxLDL into the cytosol. alpha-Tocopherol inhibits oxLDL uptake by a mechanism involving downregulation of CD36 mRNA and protein expression. Therefore, the beneficial effect of alpha-tocopherol against atherosclerosis can be explained, at least in part, by its effect of lowering the uptake of oxidized lipoproteins, with consequent reduction of foam cell formation.

Vitamin E: protective role of a Janus molecule

Since the discovery of vitamin E in 1922, its deficiency has been associated with various disorders, particularly atherosclerosis, ischemic heart disease, and the development of different types of cancer. A neurological syndrome associated with vitamin E deficiency resembling Friedreich ataxia has also been described. Whereas epidemiological studies have indicated the role of vitamin E in preventing the progression of atherosclerosis and cancer, intervention trials have produced contradictory results, indicating strong protection in some cases and no significant effect in others. Although it is commonly believed that phenolic compounds like vitamin E exert only a protective role against free radical damage, antioxidant molecules can exert other biological functions. For instance, the anti‐oxidant activity of 17‐β‐estradiol is not related to its role in determining secondary sexual characters, and the antioxidant capacity of all‐irans‐retinal is distinguished from its role in rhodopsin and vision. Thus, it is not unusual that α‐tocopherol (the most active form of vitamin E) has properties independent of its antiox‐idant/radical scavenging ability. The Roman god Janus, shown in ancient coins as having two faces in one body, inspired the designation of ‘Janus molecules’ for these substances. The new biochemical face of vitamin E was first described in 1991, with an inhibitory effect on cell proliferation and protein kinase C activity. After a decade, this nonantioxidant role of vitamin E is well established, as confirmed by authoritative studies of signal transduction and gene regulation. More recently, a tocopherol binding protein with possible receptor function has been discovered. Despite such important developments in understanding the molecular mechanism and the targets of vitamin E, its new Janus face is not fully elucidated. Greater knowledge of the molecular events related to vitamin E will help in selecting the parameters for clinical intervention studies such as population type, dose response effects, and possible synergism with other compounds.—Ricciarelli, R., Zingg, J.‐M., Azzi, A. Vitamin E: protective role of a Janus molecule. FASEB J. 15, 2314–2325 (2001)

α-Tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity

Abstract α-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of α-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive, α-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.

Vitamin E Mediates Cell Signaling and Regulation of Gene Expression

Abstract: α‐Tocopherol modulates two major signal transduction pathways centered on protein kinase C and phosphatidylinositol 3‐kinase. Changes in the activity of these key kinases are associated with changes in cell proliferation, platelet aggregation, and NADPH‐oxidase activation. Several genes are also regulated by tocopherols partly because of the effects of tocopherol on these two kinases, but also independently of them. These genes can be divided in five groups: Group 1. Genes that are involved in the uptake and degradation of tocopherols: α‐tocopherol transfer protein, cytochrome P450 (CYP3A), γ‐glutamyl‐cysteine synthetase heavy subunit, and glutathione‐S‐transferase. Group 2. Genes that are implicated with lipid uptake and atherosclerosis: CD36, SR‐BI, and SR‐AI/II. Group 3. Genes that are involved in the modulation of extracellular proteins: tropomyosin, collagen‐α‐1, MMP‐1, MMP‐19, and connective tissue growth factor. Group 4. Genes that are connected to adhesion and inflammation: E‐selectin, ICAM‐1 integrins, glycoprotein IIb, IL‐2, IL‐4, IL‐1b, and transforming growth factor‐β (TGF‐β). Group 5. Genes implicated in cell signaling and cell cycle regulation: PPAR‐γ, cyclin D1, cyclin E, Bcl2‐L1, p27, CD95 (APO‐1/Fas ligand), and 5a‐steroid reductase type 1. The transcription of p27, Bcl2, α‐tocopherol transfer protein, cytochrome P450 (CYP3A), γ‐glutamyl‐cysteine sythetase heavy subunit, tropomyosin, IL‐2, and CTGF appears to be upregulated by one or more tocopherols. All the other listed genes are downregulated. Gene regulation by tocopherols has been associated with protein kinase C because of its deactivation by α‐tocopherol and its contribution in the regulation of a number of transcription factors (NF‐κB, AP1). A direct participation of the pregnane X receptor (PXR) / retinoid X receptor (RXR) has been also shown. The antioxidant‐responsive element (ARE) and the TGF‐β‐responsive element (TGF‐β‐RE) appear in some cases to be implicated as well.

Free radical biology - terminology and critical thinking.

What is an antioxidant? Can one, at a cellular level, speak of direct and indirect antioxidants? Can oxidative stress be quantified and characterized? What are the oxidant species that may have regulatory functions in a cell? Since the above concepts have become of frequent use in all Journals it may be appropriate if some critical thinking outlined in this review could become available to a broad public.

Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols

alpha-Tocopherol, the most active form of vitamin E, causes a dose-dependent inhibition of serum-induced proliferation of smooth muscle cells (A7r5) in culture. Some tocopherol-related compounds exhibiting various degrees of antioxidant potency have also been tested on cellular proliferation. No direct correlation between the antioxidant activity of these compounds and their effect on smooth muscle cell growth could be observed. While most of the derivatives employed were not effective in inhibiting protein kinase C, in the case of alpha-tocopherol the antiproliferative effect was found to be parallel to the inhibition of protein kinase C activity, as measured in streptolysin-O permeabilized cells.

A Novel Human Tocopherol-associated Protein CLONING, IN VITRO EXPRESSION, AND CHARACTERIZATION

Vitamin E (α-tocopherol) is an essential dietary nutrient for humans and animals. The mechanisms involved in cellular regulation as well as in the preferential cellular and tissue accumulation of α-tocopherol are not yet well established. We previously reported (Stocker, A., Zimmer, S., Spycher, S. E., and Azzi, A. (1999)  IUBMB Life 48, 49–55) the identification of a novel 46-kDa tocopherol-associated protein (TAP) in the cytosol of bovine liver. Here, we describe the identification, the molecular cloning into Escherichia coli, and the in vitroexpression of the human homologue of bovine TAP,hTAP. This protein appears to belong to a family of hydrophobic ligand binding proteins, which have the CRAL (cis-retinal binding motif) sequence in common. By using a biotinylated α-tocopherol derivative and the IASys resonant mirror biosensor, the purified recombinant protein was shown to bind tocopherol at a specific binding site with K d 4.6 × 10−7 m. Northern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed. The highest amounts of hTAP message are found in liver, brain, and prostate. In conclusion, hTAP has sequence homology to proteins containing the CRAL_TRIO structural motif. TAP binds to α-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14.