(The FASEB Journal. 1999;13:965-975.)
© 1999 FASEB
Vitamin E and vascular homeostasis: implications for atherosclerosis
JOHN F. KEANEY, JR.*1,
DANIEL I. SIMON
and
JANE E. FREEDMAN
* Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, 02118, USA;
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts 02114, USA; and
Departments of Medicine and Pharmacology, Georgetown University, Washington, D.C. 20007, USA
1Correspondence: Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St., W507, Boston, MA 02118, USA. E-mail: jkeaney{at}bu edu
 |
ABSTRACT
|
|---|
Considerable epidemiologic data suggest that dietary consumption of
vitamin E reduces the incidence of cardiovascular disease. The precise
mechanisms are not clear, but emerging data indicate that vitamin E has
numerous activities that may, in part, explain its effect on vascular
disease. In particular, vitamin E enhances the bioactivity of nitric
oxide, inhibits smooth muscle proliferation, and limits platelet
aggregation. One common mechanism to account for these effects of
vitamin E is the inhibition of protein kinase C stimulation. In the
setting of atherosclerosis, inhibition of protein kinase C by vitamin E
would be expected to maintain normal vascular homeostasis and thus
reduce the clinical incidence of cardiovascular disease.—Keaney,
J. F., Jr., Simon, D. I., Freedman, J. E. Vitamin E and
vascular homeostasis: implications for atherosclerosis.
Key Words: LDL • vascular homeostasis • 
-tocopherol • nitric oxide
|
LOW DENSITY LIPOPROTEINS AND ATHEROSCLEROSIS
|
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THE CAUSAL RELATION between serum cholesterol and
atherosclerosis is now firmly established. Observations from the
Framingham Heart Study indicate that serum low density lipoprotein
(LDL)2
cholesterol is positively
associated with an increased risk of cardiovascular disease (1)
.
Intervention trials have also established that lowering LDL cholesterol
reduces the clinical manifestations of atherosclerosis (2,
3)
. Despite
this causal role for LDL cholesterol in atherosclerosis, it is worth
noting that LDL particles do not demonstrate atherogenic effects
in vitro. Macrophages incubated with very high levels of LDL
cholesterol do not become loaded with cholesterol to form foam cells,
the characteristic cell type of early atherosclerotic lesions (4,
5)
.
Endocytosis of LDL via the LDL receptor is too slow to support foam
cell formation and is also subject to down-regulation such that
cholesterol accumulation is severely limited (5,
6)
. In contrast,
modification of LDL via acetylation (5)
or oxidation (4)
facilitates
LDL recognition by a different class of receptors, known collectively
as `scavenger' receptors. Acetylation of LDL does not occur in
vivo; however, under controlled conditions, all major cell types
within the arterial wall do support LDL oxidation (4,
7
8
9)
. The
discovery that LDL oxidation is necessary for foam cell formation has
led to development of the `oxidative modification hypothesis' of
atherosclerosis (10)
.
According to this hypothesis (Fig. 1
), LDL traverses the subendothelial arterial space where it is subjected
to oxidation by adjacent vascular cells. In vitro evidence
indicates that once oxidized, LDL becomes a ligand for scavenger
receptors, leading to foam cell formation. Oxidized LDL (ox-LDL) is
also chemotactic for cultured monocytes (11)
and stimulates cellular
production of chemokines (12)
, potentially leading to inflammatory cell
recruitment into the arterial wall. In addition, ox-LDL produces both
cellular dysfunction (13)
and death (8)
, two phenomena that may further
promote the atherogenic process. Thus, LDL oxidation triggers a number
of events that can promote both the establishment and progression of
atherosclerosis.
Considerable experimental evidence now supports the oxidative
modification hypothesis. Histological studies have demonstrated the
presence of oxidized LDL in atherosclerotic lesions, but not in normal
arterial segments (14)
. LDL extracted from human or rabbit
atherosclerotic lesions has distinct physical and chemical
characteristics identical to LDL oxidized ex vivo (15)
.
Structurally unrelated antioxidant compounds such as probucol (16
17
18
19)
and N, N'-diphenyl-phenylenediamine (20,
21)
reduce
atherosclerosis in a variety of animal models. Finally, mice lacking
the scavenger receptor type A gene demonstrate a marked resistance to
atherosclerosis (22)
.
|
HUMAN STUDIES
|
|---|
Inspired by the oxidative modification hypothesis, some human
studies have examined the role of antioxidant treatment for prevention
of atherosclerotic vascular disease. From these studies we have
realized that not all antioxidants are equivalent. For example,
ß-carotene is ineffective in preventing LDL lipid peroxidation
in vitro and, consequently, clinical studies examining its
effect in cardiovascular disease have been uniformly negative (23
24
25)
.
In contrast, in ex vivo assays vitamin E inhibits the
oxidation of LDL derived from individuals who have consumed doses
greater than 25 IU/day (26)
, and emerging evidence suggests that
vitamin E may reduce clinical events associated with atherosclerosis.
Two prospective studies have demonstrated that individuals consuming
more than 100 IU/day of vitamin E experience considerably fewer
cardiovascular events than individuals who do not consume supplemental
vitamin E (27,
28)
. Two randomized, placebo-controlled trials of
vitamin E have examined its effects on cardiovascular events. In the
Alpha-Tocopherol Beta-Carotene Study, a low dose of -tocopherol
showed a modest reduction in angina and nonfatal heart attacks (29)
. At
higher doses (400–800 IU/day) of vitamin E, the Cambridge Heart
Antioxidant Study (CHAOS) found that vitamin E produced a 77%
reduction in the occurrence of myocardial infarctions and nearly a 50%
reduction in all cardiovascular events. This effect was evident as soon
as 6 months after beginning therapy, begging the question as to the
mechanism of vitamin E action.
|
REGRESSION OF ATHEROSCLEROSIS
|
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One potential mechanism for vitamin E in reducing clinical events
is the regression of atherosclerotic lesions, and there is evidence
suggesting that people on vitamin E supplements (>100 IU/day)
demonstrate less atherosclerotic lesion progression than those who do
not consume supplements (30)
. However, the magnitude of this effect is
quite small and out of proportion to the 77% reduction in clinical
events seen in the CHAOS trial, suggesting that vitamin E may have some
effect (perhaps on atherosclerotic plaque activity) other than simply
reducing lesion progression. A similar argument has been made for
cholesterol-lowering therapy (31
32
33
34)
. The purpose of this review will
be to examine actions of vitamin E that affect vascular homeostasis and
may, in part, provide a rationale for reduced vascular events in the
absence of profound angiographic changes in atherosclerotic lesions.
|
VASCULAR HOMEOSTASIS
|
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Normal organ perfusion requires a stable local environment within
the vascular wall. Smooth muscle cells must be maintained in a
quiescent state, platelet adhesion and blood coagulation must be
inhibited, and the regulation of vascular tone requires precise
modulation. Many of these homeostatic functions are served by the
vascular endothelium, which acts as a local integrator of paracrine and
autocrine signals. One major factor responsible for vascular
homeostasis is nitric oxide (•NO), a free
radical species synthesized by a family of enzymes, termed the nitric
oxide synthases (NOS). Endothelial elaboration of
•NO is known to control vascular tone (35)
,
smooth muscle cell phenotype (36,
37)
, and the adhesion/aggregation of
leukocytes and platelets (38
39
40
41
42)
. In mice lacking the endothelial
isoform of NOS (eNOS), spontaneous hypertension (43,
44)
and defective
vascular remodeling (45)
have been observed. In patients with impaired
plasma •NO bioactivity, spontaneous arterial
thrombosis ensues (46)
. Taken together, available evidence indicates
that •NO is important in the control of
vascular homeostasis.
There is a growing consensus that improving •NO
bioactivity in patients with atherosclerosis is associated with a
reduction in cardiovascular events. Evidence to support this contention
is derived mainly from studies demonstrating that therapies known to
reduce the risk of vascular events in atherosclerosis also improve
indices of •NO bioactivity. For example,
postmenopausal women receiving hormonal replacement therapy tend to
have fewer vascular events than women without hormonal replacement (47,
48)
, and estrogen replacement in such women also improves
•NO bioactivity (49)
. Studies have demonstrated
that cholesterol lowering with HMG-CoA reductase inhibitors reduces
cardiovascular events (50,
51)
; such treatment is associated with
improved •NO-mediated arterial relaxation in
the coronary arteries as well (52)
. Thus, by extension, one might
predict that any treatment resulting in enhanced
•NO bioactivity would also favorably affect
vascular events in patients with atherosclerosis.
|
VITAMIN E AND ENDOTHELIUM- DERIVED •NO
|
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The bioactivity of •NO is particularly
sensitive to oxidative stress. Superoxide combines readily with
•NO in a diffusion-limited reaction
(k=1.9x1010
M-1 · s-1) (53)
to
form peroxynitrite (54)
, a compound with considerably less bioactivity
than •NO itself (55)
. In atherosclerosis (56)
,
hypercholesterolemia (57)
, hypertension (58)
, and diabetes (59)
, the
superoxide flux within the vascular wall is often increased, leading to
reduced •NO action. Moreover, peroxynitrite is
a potent oxidant (54)
and the formation of peroxynitrite leads to lipid
peroxidation (60)
, which is known to impair •NO
bioactivity (61
62
63)
.
Since •NO is an important component of vascular
homeostasis and its action is sensitive to oxidative stress, it is not
surprising that vitamin E may have some effect on
•NO bioactivity. One well-described model of
impaired •NO bioactivity is the cholesterol-fed
rabbit (64)
. In this model, a high-cholesterol diet for 4 to 10 wk
produces a progressive impairment of
•NO-mediated arterial relaxation that is
characterized by increased oxidative stress (56,
57,
65
66
67)
. Treatment
of cholesterol-fed rabbits with -tocopherol both inhibits LDL
oxidation and preserves •NO bioactivity
(68
69
70)
. Since normal vessels exposed to ox-LDL demonstrate impaired
•NO bioactivity (13)
, one might speculate that
inhibition of LDL oxidation by -tocopherol is the mechanism for
preserved •NO bioactivity. However, inhibition
of LDL oxidation alone in this model is not sufficient to preserve
•NO bioactivity, as animals consuming very high
doses of -tocopherol (250-fold nutritional excess) actually
demonstrate worse •NO-mediated arterial
relaxation than animals fed cholesterol alone despite considerable
inhibition of LDL oxidation (71)
. Thus, these data point to some other
activity of -tocopherol within the vascular wall that leads to
preserved endothelium-derived •NO action.
-Tocopherol localizes mainly to lipoproteins and membranes, where it
serves to scavenge lipid peroxyl radicals (72)
. Despite this activity,
atherosclerosis is characterized by lipid peroxidation within the
vascular wall even in the presence of -tocopherol (73)
. In light of
these findings, it is possible that
•NO-mediated arterial relaxation is preserved
by -tocopherol independent of its effect to inhibit lipid
peroxidation. This possibility was investigated by manipulating the
vascular content of -tocopherol in rabbits by dietary means (74)
.
Depleting the diet of total tocopherols resulted in an aortic
-tocopherol content of 0.4 nmol/g, whereas supplementation of the
diet with -tocopherol produced a corresponding level of 63 nmol/g.
This difference in vascular -tocopherol content resulted in
strikingly different sensitivities to ox-LDL. Arteries deficient in
-tocopherol demonstrated a dose-dependent impairment of
•NO-mediated arterial relaxation upon exposure
to ox-LDL (74)
. In contrast, vessels containing abundant -tocopherol
were markedly resistant to this effect of ox-LDL. These data suggest
that vascular -tocopherol content is an important determinant of the
vascular response to ox-LDL. Further investigation of this phenomenon
revealed that -tocopherol had no bearing on ox-LDL-induced
cytotoxicity or endothelial denudation (74)
. Rather, vascular
-tocopherol prevented protein kinase C stimulation that was due to
ox-LDL (Fig. 2
) (74)
and was responsible for the impairment in
•NO bioactivity (75)
. Thus, inhibition of
protein kinase C stimulation by -tocopherol has important
implications for vascular function in atherosclerosis.
|
HUMAN STUDIES OF VITAMIN E AND •NO BIOACTIVITY
|
|---|
Clinical studies in humans on the role of vitamin E in preserving
•NO bioactivity are not as well developed as
animal studies. Three studies involving patients with either
hypercholesterolemia (76,
77)
or myocardial infarction (78)
have
examined the effect of vitamin E on vascular function. These studies
have been uniformly negative for any effect on
•NO bioactivity, and the reasons for this are
not entirely clear. Possible explanations for this discrepancy include
the duration of treatment or the dose of antioxidants, both of which
have generally been greater in animal studies.
The index of •NO bioactivity used in human
studies is another important consideration. Typically, animal studies
have focused on •NO bioactivity in conduit
blood vessels, whereas the aforementioned human studies have examined
the action of •NO in resistance arterioles.
Recent reports involving vitamin E treatment and
•NO-mediated, endothelium-dependent conduit
vessel function are of particular importance in this regard. Koh and
colleagues studied postmenopausal women treated with either
-tocopherol (800 IU/day) or estrogen replacement (79)
. After 6 wk,
-tocopherol treatment produced a significant improvement in
•NO-mediated arterial relaxation
measured as flow-mediated dilation of the brachial artery (79)
. The
extent to which •NO-mediated arterial
relaxation improved with -tocopherol was similar to that seen with
estrogen replacement. Indices of oxidation were not determined in that
study. Patients with high remnant (triglyceride rich) lipoprotein
levels demonstrate impaired •NO-mediated
arterial relaxation in the coronary arteries (80)
. Motoyama and
colleagues (81)
have demonstrated that -tocopherol supplementation
in these patients improves •NO-dependent,
flow-mediated dilation of the brachial artery. These findings are also
consistent with recent observations from patients with coronary spastic
angina demonstrating that abnormal •NO-mediated
brachial artery relaxation is normalized by -tocopherol treatment
(82)
. The effect of vitamin E on •NO-mediated
arterial relaxation appears to be additive with cholesterol-lowering
therapy (83)
, as is the case with probucol, another lipid-soluble
antioxidant (84)
.
|
VITAMIN E AND SMOOTH MUSCLE PROLIFERATION
|
|---|
One hallmark of the atherosclerotic plaque is proliferation of
vascular smooth muscle. Smooth muscle cells that migrate into the
vessel intima are the principal type of cell within the matrix of the
atherosclerotic plaque. Patients who demonstrate rapid lesion
progression also exhibit excess cardiovascular events (85)
, suggesting
that the rate of smooth muscle proliferation in lesions is an important
determinant of lesion stability. One might expect agents that inhibit
smooth muscle cell proliferation to reduce lesion progression and thus
limit clinical vascular events. Considerable in vitro data
indicate that -tocopherol inhibits the proliferation of smooth
muscle cells. Using the A7r5 smooth muscle cell line, Boscoboinik and
colleagues demonstrated that physiological concentrations of
-tocopherol (50 µM) inhibited
[3H]-thymidine incorporation in response to
serum, PDGF-BB, and endothelin (86,
87)
. Inhibition of cell
proliferation in the A7r5 cells was associated with a reduction in
protein kinase C activity (86,
87)
, suggesting that -tocopherol
inhibits smooth muscle cell proliferation principally as a consequence
of protein kinase C inhibition. A similar finding has been observed for
cellular proliferation stimulated by either LDL or
malondialdehyde-modified LDL (88)
. Calphostin C, a specific protein
kinase C inhibitor, also inhibits smooth muscle cell proliferation,
further supporting the notion that -tocopherol acts through the
inhibition of protein kinase C stimulation (89)
. It should be noted,
however, that the data on vitamin E inhibition of smooth muscle cell
proliferation is based almost entirely on in vitro data, and
experience in vivo has thus far been mixed (90,
91)
.
|
VITAMIN E AND PLATELET FUNCTION
|
|---|
The inciting event for cardiovascular disease is typically
thrombus formation within the vessel lumen (92)
, often precipitated by
the adhesion and aggregation of platelets to a ruptured atherosclerotic
plaque (93)
. Consistent with this idea, plasma levels of
platelet-derived thromboxane and prostaglandin metabolites are
increased in patients with acute coronary syndromes (94)
; aspirin
treatment has uniformly been associated with a reduced risk of
thrombotic complications from atherosclerosis (95)
. Any activity of
-tocopherol to inhibit platelet function would therefore be expected
to reduce vascular events in patients with atherosclerosis.
Higashi and Kikuchi (96)
were the first to demonstrate that
-tocopherol inhibits the aggregation of platelets by using hydrogen
peroxide as the aggregatory stimulus. Subsequent studies by Steiner and
Anastasi (97)
demonstrated that -tocopherol also inhibited platelet
aggregation in response to epinephrine, collagen, and ATP. These early
studies with -tocopherol were plagued by the need for
supraphysiological levels of -tocopherol for meaningful inhibition
of platelet aggregation (0.3 to 2.0 mM) (97)
. Plasma -tocopherol
concentrations typically range from 15 to 40 µM (98)
in normal
individuals, increasing up to threefold with oral supplementation (99)
.
So it seemed questionable that -tocopherol would have any effect on
platelets at physiological concentrations. However, it is now apparent
that pharmacologic concentrations of -tocopherol are needed ex
vivo in order to load platelets with -tocopherol levels
comparable to that achieved with oral supplementation (100)
.
Considerable effort has been directed at identifying the mechanism
responsible for vitamin E inhibition of platelet aggregation. A variety
of proposed mechanisms including inhibition of lipid peroxidation (97)
and cyclooxygenase inhibition (101,
102)
, have not been borne out by
experimental evidence. Until recently, the only mechanism attributed to
-tocopherol was the inhibition of pseudopodia formation on platelet
contact with fibrinogen-coated glass slides (103)
. More recent studies
have begun to shed light on this difficult problem. We have observed
that platelet aggregation by phorbol ester is particularly sensitive to
-tocopherol, whereas ADP-induced platelet aggregation is rather
insensitive (100)
. Such findings implicate protein kinase C as a target
for the effect of vitamin E on platelet aggregation (104)
. This
suspicion has been borne out by experimental evidence indicating that
-tocopherol potently inhibits protein kinase C stimulation in human
platelets (Fig. 3
) (100)
, a finding also confirmed recently in megakaryoblastic cells
(105)
.
Inhibition of protein kinase C stimulation provides an attractive
mechanism for the effects of -tocopherol on platelet aggregation,
particularly in light of previous studies. Early trials of oral
-tocopherol supplementation failed to demonstrate any significant
effect on platelet aggregation (97,
106)
. Such findings are readily
explained by the agonists used to stimulate platelet aggregation. These
studies used ADP, epinephrine, and collagen at concentrations that
largely affect platelet aggregation in a protein kinase C-independent
manner (104)
. Thus, one might now predict that -tocopherol would
have no affect under such conditions. The aforementioned effects of
-tocopherol on shear-induced platelet pseudopodia formation (103)
are also of interest. Shear stress is now known to evoke protein kinase
C stimulation in platelets (107)
, and the adherence of platelets to
fibrinogen is critically dependent on protein kinase C activity (108)
.
Thus, one can reconcile some previously disparate findings with the
current knowledge that -tocopherol inhibits platelet function
through a protein kinase C-dependent mechanism (100)
.
The implications of platelet -tocopherol for platelet function are
not limited solely to the aggregation response. For example, platelets
contain NOS, which results in the production of
•NO late during the aggregation response (41,
42)
and has important implications for vascular homeostasis. Platelets
derived from mice lacking the eNOS gene release no measurable
•NO and exhibit bleeding times that are 43%
shorter than those of wild-type mice (109)
. We have recently found that
loading platelets with physiologically relevant levels of vitamin E is
associated with a dose-dependent increase in platelet
•NO production during aggregation (110)
. Acute
physiological loading of platelets with -tocopherol increased
platelet •NO production 158% and reduced the
extent of aggregation from 84% to 58% (P<0.01). The
precise mechanisms responsible for this effect are still under
investigation, although platelet -tocopherol status appears to be
important in regulating the amount of superoxide elaborated during
aggregation (110)
. In -tocopherol-loaded platelets, superoxide
release is 74% less than that of control platelets (110)
. Thus,
reducing platelet superoxide production during aggregation is one
potential mechanism for increased •NO with
-tocopherol. Alternatively, protein kinase C stimulation in
endothelial cells impairs receptor-mediated •NO
production (111)
, and one might speculate that similar mechanisms are
operative in platelets.
It is likely that inhibition of protein kinase C stimulation by
-tocopherol will have important implications in developing platelets
as well. In cultured endothelial cells, inhibition of protein kinase C
is associated with an up-regulation of eNOS transcription (112)
and an
increase in endothelium-derived •NO. We have
recently found that inhibition of protein kinase C in immortalized
megakaryocytes is also associated with an up-regulation of eNOS message
and protein. It is likely this effect is physiologically relevant, as
circulating platelet vitamin E status has a direct bearing on platelet
•NO production. In 87 consecutive patients
undergoing coronary angiography, platelet
•NO production correlated with plasma
-tocopherol concentration (R=0.5; P<0.01), and this
effect was independent of aspirin and nitrate treatment (113)
.
Thus, it appears that platelet vitamin E content is an important
determinant in platelet responsiveness toward protein kinase
C-dependent stimuli. Considering that platelets play a critical role in
the acute manifestations of atherosclerotic vascular disease, it is
plausible to speculate that any effect of vitamin E on cardiovascular
disease could well be mediated through a modulation of platelet
function. Recent data indicating that patients treated with vitamin E
demonstrated an excess risk for hemorrhagic stroke would tend to
support this contention (25)
.
|
VITAMIN E AND MONOCYTE FUNCTION
|
|---|
There is now a growing appreciation that atherosclerosis is an
inflammatory disease and that the extent of inflammation is related to
disease activity. Inflammatory markers such as fibrinogen and
C-reactive protein correlate with the clinical manifestations of
atherosclerosis (114,
115)
. These clinical findings are consistent with
histological studies demonstrating that atherosclerotic plaque rupture
occurs at sites characterized by a large population of inflammatory
cells (34)
. Taken together, these findings suggest that the
inflammatory environment contributes significantly to atherosclerotic
plaque rupture and, thus, the clinical manifestations of
atherosclerosis. The effect of -tocopherol to alter the inflammatory
environment of the vascular wall may have important implications for
clinical vascular events.
The recruitment of inflammatory cells into the vascular wall is an
important component of vascular inflammation. The regulation of this
process can occur at either the level of the endothelium and/or the
leukocyte. With respect to the former, endothelial cells loaded with
-tocopherol demonstrate a reduction in agonist-induced monocyte
adhesion (116)
. In particular, the endothelial cell -tocopherol
content regulates the surface expression of E-selectin in response to
IL-1ß, thereby reducing monocyte adhesion to endothelial cells (116)
.
The precise mechanism for these observations is not yet clear. Specific
antioxidants such as probucol and N-acetylcysteine produce similar
effects in endothelial cells, whereas other antioxidants do not. Thus,
with respect to the endothelial cell, -tocopherol regulates monocyte
adhesion through a reduction in surface expression of E-selectin.
-Tocopherol also has effects specific to the monocyte. Devaraj and
colleagues examined monocytes isolated from patients supplemented with
1200 IU/day of -tocopherol over 8 wk and demonstrated that reactive
oxygen species release in response to IL-1ß was reduced compared with
placebo (117)
. Monocytes from -tocopherol-supplemented patients were
also less able to oxidize lipids or adhere to an activated endothelial
cell monolayer (117)
. A similar phenotype was observed in monocytes
treated with the specific protein kinase C inhibitor, calphostin C (117)
.
The mechanisms for such observations have been explored in our
laboratory. We have examined the effect of -tocopherol on
inflammatory cell adhesion, using the monocytic U937 cell line. We
observed dose- and time-dependent incorporation of -tocopherol into
U937 cells that resulted in significant inhibition of adhesion to
purified fibrinogen and intercellular adhesion molecule-1 (ICAM-1), but
not fibronectin, thereby implicating -tocopherol as a modulator of
ß2 integrin function (118)
. Several
ß2 integrins are expressed in U937 cells,
including LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), and p150,95
(CD11c/CD18). To more precisely identify leukocyte integrins regulated
by -tocopherol, we transfected erythroleukemic K562 cells, which
normally lack ß2 integrins, with Mac-1. We
found that -tocopherol blocked the adhesion of Mac-1 transfected
K562 cells to fibrinogen and ICAM-1 (118)
, implicating Mac-1 as an
integrin target for -tocopherol (118)
. Thus, mononuclear cell
incorporation of -tocopherol over the physiological range is
associated with inhibition of ß2
integrin-dependent cellular adhesion. The relative importance of
protein kinase C inhibition or antioxidant action by
-tocopherol is the focus of ongoing studies.
|
MATTERS OF CONTROVERSY
|
|---|
A recurrent aspect of the discussion outlined above is the
activity of -tocopherol to inhibit protein kinase C stimulation.
However, the precise mechanism responsible for this effect remains a
matter of considerable debate. The first report that vitamin E
inhibited protein kinase C activity involved incubation of semipurified
enzyme preparations with -tocopherol and suggested a direct
inhibitory effect on the enzyme with an IC50 of
either 450 µM (119)
or ~25 µM (Fig. 4
) (87)
. However, subsequent studies using purified enzyme preparations
have yielded different results. Kunisaki and colleagues found that
-tocopherol at concentrations of up to 400 µg/ml (~930 µM) had
no direct effect on the activity of protein kinase C isoforms and
ßII (120)
. Unpublished studies from our own
laboratory also indicate that -tocopherol (up to 1% of
phospholipid) has no direct effect on diacylglycerol- or
calcium-stimulated protein kinase C or ßII
activity. Thus, direct enzymatic inhibition of protein kinase C does
not appear a likely mechanism for -tocopherol to inhibit stimulation
of the enzyme.
In evaluating potential mechanisms for -tocopherol inhibition of
protein kinase C stimulation, one must consider specific isoforms of
the enzyme. Protein kinase C has 11 known isoforms, which are divided
into three distinct groups: classical, novel, and atypical (121)
. Of
these, the classical (, ßI,
ßII, 
) isoforms have been studied in the
greatest detail with respect to -tocopherol. Activation of classical
protein kinase C isoforms is dependent on enzyme translocation from the
cytosol to the membrane (121)
. Since -tocopherol is lipid soluble,
inhibition of translocation represents a potential means of regulation.
Boscoboinik and colleagues found that smooth muscle cells treated with
-tocopherol exhibited a shift of total protein kinase C from the
membrane to the cytosol, and suggested that inhibition of translocation
was responsible for the action of -tocopherol (86,
87)
. This notion
is supported by recent findings in megakaryoblastic cells demonstrating
that protein kinase C translocation in response to phorbol ester is
inhibited by -tocopherol (105)
. However, not all studies have
demonstrated that -tocopherol inhibits protein kinase C
translocation. Kunasaki and colleagues found that -tocopherol had no
effect on phorbol ester-stimulated translocation of protein kinase C
or ßII in rat aortic smooth muscle cells
(122)
. In our own laboratory, we have found no effect of -tocopherol
on protein kinase C or ßII translocation in
phorbol ester-stimulated human platelets (unpublished observations).
Thus, any consensus concerning the effect of -tocopherol on protein
kinase C translocation requires further studies that use a variety of
cell types and more diverse stimuli for protein kinase C activation
other than just phorbol ester.
Considerable data support an indirect mechanism for the effect of
-tocopherol on protein kinase C stimulation. For example,
hyperglycemia is known to activate protein kinase C
ßII in smooth muscle cells through an increase
in de novo diacylglycerol synthesis, and -tocopherol
abrogates the effect of hyperglycemia by decreasing cellular
diacylglycerol levels (120,
122)
through stimulation of DAG kinase
activity (123)
. With respect to smooth muscle cell proliferation, the
antiproliferative effect of -tocopherol is prevented by okadaic
acid, suggesting that protein kinase C phosphorylation status and/or
protein phosphatase activity is involved in the action of
-tocopherol (89)
. Clearly, further investigation will be needed to
determine the precise target(s) of -tocopherol that mediate its
activity to prevent protein kinase C stimulation.
Despite all the controversy surrounding the mechanism of -tocopherol
action, certain aspects of its protein kinase C inhibitory capacity are
clear. In particular, the antioxidant activity of -tocopherol is not
required for its activity against protein kinase C stimulation.
Tasinato and colleagues have demonstrated that ß-tocopherol, an
isoform of -tocopherol, has no activity with regard to smooth muscle
cell proliferation and inhibition of protein kinase C stimulation (89)
.
Since ß-tocopherol accumulates within cells to a similar extent as
-tocopherol and exhibits ~52% of its antioxidant activity (72)
, a
compelling argument can be made that antioxidant activity alone cannot
mediate protein kinase C inhibition (88,
89,
124)
. Data from our
laboratory would agree with this position. We have found that both
-tocopheryl acetate and -tocopheryl quinone, two forms of vitamin
E without antioxidant activity, inhibit protein kinase C stimulation by
phorbol ester (unpublished observations). More investigation will be
needed to precisely define the structure/activity correlates of
-tocopherol with respect to the inhibition of protein kinase C
stimulation.
|
UNANSWERED QUESTIONS
|
|---|
Although there is general consensus regarding the activity of
-tocopherol to inhibit protein kinase C stimulation, important
questions have yet to be resolved. For example, the precise isoforms of
protein kinase C that are sensitive to -tocopherol remain to be
determined. Early data suggests that protein kinase C may be a
target (124)
, but definitive data will probably require standardized
isoform-specific protein kinase C activity assays. Similarly, the
precise vitamin E structural requirements for inhibition of protein
kinase C stimulation are still unknown. Finally, the specific
mechanisms whereby -tocopherol renders protein kinase C insensitive
to stimulation, perhaps through some change in protein kinase C
phosphorylation status (124)
, remain to be determined. Answers to these
questions should help to shed light on the regulation of protein kinase
C and provide important insights into the treatment of vascular
diseases.
|
CONCLUSIONS
|
|---|
It is clear that atherosclerotic vascular disease, and risk
factors predisposing to atherosclerosis, are characterized by a state
of heightened oxidative stress within the vascular wall. One important
component of this oxidative stress is the oxidative modification of LDL
that forms the basis for the oxidative modification hypothesis of
atherosclerosis (10)
. Although this model accurately predicts many
early events associated with atherosclerosis, we now know that
oxidative stress within the vascular wall is a complex phenomenon
involving a variety of lipid- and water-soluble sources of oxidative
damage (33)
. Epidemiologic data suggest that antioxidant
supplementation may be associated with a reduced risk of clinical
events from atherosclerosis; however, interventional trials only
support a role for vitamin E in this regard. What properties of vitamin
E can account for such a distinction? Unlike many other antioxidants,
vitamin E demonstrates a broad range of effects that promote vascular
homeostasis. One important property of vitamin E that may be
responsible for such a distinction is its capacity to inhibit protein
kinase C stimulation. Only continued investigation into the
mechanism(s) of vitamin E action will ultimately determine whether it
holds promise as a therapeutic intervention for patients with vascular
disease.
|
ACKNOWLEDGMENTS
|
|---|
The authors are supported by grants from the American Heart
Association and the National Institutes of Health. J.F.K. is the
recipient of a Clinical Investigator Development Award (HL03195) and
J.E.F. is the recipient of a Mentored Clinical Scientist Development
Award (HL03556), both from the National Institutes of Health.
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FOOTNOTES
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2 Abbreviations: CHAOS, Cambridge Heart Antioxidant Study;
eNOS, endothelial isoform of nitric oxide synthase; ICAM-1,
intercellular adhesion molecule-1; LDL, low density lipoprotein;
ox-LDL, oxidized LDL; PMA, phorbol ester; •NO, nitric
oxide; NOS, nitric oxide synthases. 
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ABSTRACT
LOW DENSITY LIPOPROTEINS AND...
,
macrophages. Adapted from ref 11
