Vitamin E and atherosclerosis

Background information can be found in Section
4.5.
Vitamin E counteracts many of the atherogenic
effects of oxidized LDL (listed in Section 4.5.3) by
mechanisms that may be independent of its antioxidant
properties. As discussed in this section, some
mechanisms are attributable to an inhibitory effect of
vitamin E on protein kinase C (PKC) activity.
9.7.1 Inhibition of protein kinase C activity
Physiological concentrations of α-tocopherol markedly
inhibit PKC activity in vascular smooth muscle
cells (Azzi & Stocker, 2000). Inhibition is obtained
only at the cellular level; addition of α-tocopherol to
recombinant PKC in vitro does not result in inhibition.
β-Tocopherol, which possesses 89% of the antioxidant
potency of α-tocopherol, is not inhibitory; however, it
is able to reverse the inhibitory effect of α-tocopherol.
Other tocochromanols (γ- and δ-tocopherols and α-
and γ-tocotrienols) are also not inhibitory (Chatelain
et al., 1993). Thus the inhibitory effect of vitamin E on
PKC activity is specifi c to α-tocopherol and is apparently
unrelated to its antioxidant activity. Although
various isoforms of PKC (α, β, δ, ε, ζ and μ) have been
shown to be present in rat aortic smooth muscle cells,
only PKCα is inhibited by α-tocopherol (Ricciarelli et
al., 1998). The inhibition is indirect and not attributable
to a decreased synthesis of the enzyme. There is
evidence that α-tocopherol induces the activity of a
type 2A phosphatase (Ricciarelli et al., 1998), an enzyme
which desensitizes the PKC signalling pathway
242 Vitamins: their role in the human body
by dephosphorylating PKCα (Hansra et al., 1996).
Whether or not the type 2A phosphatase is the only
target of α-tocopherol is under investigation.
9.7.2 Protection of low-density lipoprotein
from oxidation
Several reports indicate protection of LDL from
oxidation following supplementation of human diets
with α-tocopherol (Dieber-Rotheneder et al., 1991;
Jialal & Grundy, 1992; Princen et al., 1992; Reaven
et al., 1993; Jialal et al., 1995). Supplementation can
increase the vitamin E content of LDL to about four
times its basal level (Esterbauer et al., 1990). In one
report (Reaven et al., 1993), dietary supplementation
with 1600 mg all-rac-α-tocopherol per day (1760 IU
per day) for 5 months resulted in a 2.5-fold increase in
LDL vitamin E levels and a 50% decrease in LDL susceptibility
to oxidation as measured by in vitro assays.
Jialal et al. (1995) showed that the minimum dose of
α-tocopherol needed to signifi cantly decrease the susceptibility
of LDL to oxidation was 400 IU per day.
The release of superoxide by phagocytic monocytes
during the respiratory burst (Section 5.2.3) induces
oxidation of LDL and renders it toxic to proliferating
cells (Cathcart et al., 1989). Monocytes from healthy
human subjects taking oral vitamin E supplements
(1200 IU per day) showed lower superoxide production
and a reduced capacity to oxidize LDL (Devaraj
et al., 1996). This effect of vitamin E appeared to be
mediated via inhibition of PKC. Vitamin E may therefore
protect circulating LDL from oxidation induced
by activated phagocytes. The enzyme responsible for
superoxide production in phagocytes is NADPH-oxidase.
Activation of this enzyme, elicited by appropriate
stimulation of the phagocytic cell, requires translocation
of several cytosolic enzyme components to
the membrane. PKC is involved in the activation of
NADPH-oxidase and can phosphorylate one of its
cytosolic components, p47phox. Cachia et al. (1998a)
studied the effect of vitamin E on NADPH-oxidase
activation elicited by phorbol myristate acetate in
human monocytes. They found that α-tocopherol
inhibited translocation and phosphorylation of
p47phox. The results suggested that the attenuating effect
of α-tocopherol on the respiratory burst is due to
inhibition of PKC activity.
The lysolecithin that accumulates in oxidized LDL
increases production of superoxide anion in the
walls of blood vessels, which may further enhance
LDL oxidation (Ohara et al., 1994). When human
monocytes were stimulated by phorbol ester to produce
superoxide in vitro, the addition of native LDL
inhibited superoxide production in a manner highly
sensitive to the increasing α-tocopherol content; the
free form of α-tocopherol produced lower inhibition
compared with the lipoprotein-associated form
(Cachia et al., 1998b). It was suggested that a vitamin
E-induced decrease in monocyte superoxide production
could lead to a decrease in lysolecithin production
in LDL. Lysolecithin is responsible for many of
the atherogenic properties of oxidized LDL and any
means of reducing its production would promote an
anti-atherogenic status of vessels.
9.7.3 Prevention of monocyte
transmigration
Incubation of co-cultures of human aortic endothelial
and smooth muscle cells with LDL in the presence
of human serum resulted in an increased synthesis of
monocyte chemotactic protein 1 (MCP-1) mRNA
and protein. This was accompanied by an increase in
the adhesion of monocytes (but not neutrophil-like
cells) to the endothelial monolayer and an increased
transmigration of monocytes into the subendothelial
space. The increase in monocyte migration was most
likely due to the increased levels of MCP-1, since it was
completely blocked by a specifi c antibody to MCP-1.
Pre-treatment of the co-cultures with α-tocopherol
before the addition of LDL prevented the LDL-induced
monocyte transmigration (Navab et al., 1991).
9.7.4 Inhibition of monocyte–endothelial
cell adhesion
The induced expression of the endothelial adhesion
molecules, ICAM-1, VCAM-1 and E-selectin, is a
key event in the pathogenesis of atherosclerosis. The
genetic expression of protein molecules is regulated
by transcription factors which, when activated, bind
to specifi c regulatory elements on the DNA of target
genes where they mediate gene transcription and
synthesis of the encoded protein. Expression of genes
involved in early defence reactions, such as the genes
for cytokines and cytokine receptors, endothelial and
leucocyte adhesion molecules, and some growth and
differentiation factors, depends upon a particular
Vitamin E 243
transcription factor, nuclear factor-κB (NF-κB).
NF-κB is found in many different cell types and tissues,
but has been characterized best in cells of the
immune system, such as lymphocytes, monocytes and
macrophages.
In the absence of a stimulus, NF-κB resides in the
cytoplasm as an inactive complex composed of three
subunits – two DNA-binding subunits (p65 and p50)
and an inhibitory subunit called 1κB. Various extracellular
activators cause an alteration in 1κB, allowing
it to be released from the complex. The NF-κB dimer
then migrates to the nucleus where it binds to the
DNA recognition site.
The cytoplasmic NF-κB–1κB complex is activated
by a great variety of agents. These include the cytokines
IL-1 and TNF-α, viruses, double-stranded
RNA, bacterial lipopolysaccharide (LPS), endotoxins,
T-cell mitogens, phorbol 12-myristate 13-acetate
(PMA), protein synthesis inhibitors (e.g. cycloheximide)
and UV radiation (Schreck et al., 1992).
Schreck et al. (1991) reported that treatment of
T lymphocytes with micromolar concentrations of
hydrogen peroxide activated NF-κB; that is, hydrogen
peroxide induced the nuclear appearance and DNAbinding
of the transcription factor. Hydrogen peroxide
also induced the expression of the HIV-1 provirus,
whose gene is controlled by NF-κB. The activation of
NF-κB by hydrogen peroxide was inhibited by the
antioxidant and free radical scavenger N-acetyl-Lcysteine
(NAC). These experiments strongly supported
the preconceived idea that oxygen free radicals
were involved in the activation process. After its passive
diffusion through the cell plasma membrane, the
relatively innocuous hydrogen peroxide can be converted
into the highly reactive hydroxyl radical (Section
4.3.1). Activation of NF-κB by cycloheximide,
double-stranded RNA, IL-1 and LPS (Schreck et al.,
1991) and TNF-α and PMA (Staal et al., 1990) was
also inhibited by NAC.
Every type of cell produces oxygen radicals constitutively.
It is well established that different cell types
are stimulated to enhance the production of oxygen
radicals by the binding of extracellular cytokines such
as TNF-α and IL-1 to their respective cell surface receptors.
Since these cytokines and other agents, and
also hydrogen peroxide (a free radical precursor),
are able to activate NF-κB, and all of these activators
can be inhibited by a radical-scavenging antioxidant,
Schreck & Baeuerle (1991) postulated that oxygen
radicals act as second messengers in relaying extracellular
signals to the cytosolic NF-κB–1κB complex.
Oxygen radicals are well suited for this purpose; they
are small, diffusible and ubiquitous, and can be synthesized
and destroyed rapidly. The oxygen radicals
somehow activate NF-κB, which then migrates to the
nucleus and binds to its transcription site on the DNA
(Fig. 9.3).
Faruqi et al. (1994) observed that agonist-induced
adhesion of monocytes to cultured human umbilical
vein endothelial cells was inhibited by prior treatment
with α-tocopherol. The inhibition correlated with a
decrease in steady-state levels of E-selectin mRNA
and cell surface expression of E-selectin. Probucol and
NAC were also inhibitory, whereas other antioxidants
had no signifi cant effect. PKC did not appear to play
a role in the α-tocopherol effect since no suppression
of phosphorylation of PKC substrates was observed.
Cominacini et al. (1997) showed that expression of
ICAM-1 and VCAM-1 induced by oxidized LDL
could be reduced by pre-treatment of either the
oxLDL or the endothelial cells with vitamin E. Martin
et al. (1997) demonstrated an inhibitory effect of
α-tocopherol upon LDL-induced adhesion of monocytes
to human aortic endothelial cells and an accompanying
decrease in the release of ICAM-1. Devaraj
et al. (1996) reported that monocytes isolated from
healthy human subjects supplemented with 1200 IU
per day of α-tocopherol over 8 weeks were less able,
when activated, to adhere to activated endothelial cells
compared with monocytes isolated from placebo controls.
The vitamin E-enriched monocytes also showed
a 90% decrease in the release of interleukin 1β (IL-1β)
when activated. IL-1β is a proatherogenic, proinfl ammatory
cytokine that promotes monocyte–endothelial
cell adhesion; it also augments smooth muscle cell
proliferation via induction of platelet-derived growth
factor. Enrichment of monocytes with α-tocopherol
resulted in a reduced expression of the monocyte
adhesion molecules MAC-1 and VLA-4 (Islam et al.,
1998). Furthermore, pre-treatment of monocytes
with α-tocopherol signifi cantly decreased the LPSinduced
activation of NF-κB. The results of these and
other experiments suggest that α-tocopherol inhibits
transcription of adhesion molecule genes by preventing
the activation of NF-κB by oxygen radicals generated
within the cell.