From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0682
The fact that low density lipoprotein
(LDL)1 is extremely
susceptible to oxidative damage has been known for some time (1, 2),
but until quite recently this was primarily a nuisance for the student
of lipoprotein metabolism. It now appears that oxidation of LDL plays a
significant role in atherogenesis.
Beginning in the 1980s evidence began to accumulate that cholesterol
accumulation in the developing atherosclerotic lesion was probably not
due to the uptake of native LDL by way of the Brown/Goldstein LDL
receptor but instead due to the uptake of some modified form of LDL
(then still unidentified) by way of one or more alternative receptors
(also then unidentified). This conclusion grew from two well accepted
observations. First, patients and animals totally lacking the LDL
receptor nevertheless accumulate cholesterol in foam cells much the
same way as do patients and animals with normal LDL receptors; second,
the two cell types in lesions that give rise to cholesterol-laden foam
cells (the monocyte/macrophage and the smooth muscle cell) do not
accumulate cholesterol in vitro even in the presence of very
high concentrations of native LDL (3, 4). This paradox could be
resolved if circulating LDL underwent some form of modification and if
the modified form, rather than native LDL itself, then served as the ligand for delivery of cholesterol to developing foam cells.
Acetylation of LDL in vitro generated a modified LDL that
could induce cholesterol accumulation in macrophages (3). The uptake of
this acetylated LDL was by way of a new receptor designated the
acetyl LDL receptor (later cloned and renamed scavenger
receptor A (SRA) (5). SRA, unlike the LDL receptor, is not
down-regulated when the cholesterol content of the cell increases.
Thus, acetyl LDL could, in principle, account for foam cell formation.
However, there was (and still is) no evidence that acetylation of LDL
occurs to any extent in vivo. Another modified form of LDL
emerged as a candidate when it was shown that simply incubating LDL
overnight with a monolayer of arterial endothelial cells converted it
to a form that was taken up much more rapidly by macrophages and
capable of increasing their cellular cholesterol content (6-8). The
uptake was specific and saturable, and it occurred in part by way of
the acetyl LDL receptor. Incubation with smooth muscle cells could also
modify LDL in much the same way (7, 8). This cell-mediated modification turned out to be, very simply, oxidative modification (9, 10). The
addition of antioxidants to the culture medium completely blocked
cell-induced modification, and the changes induced by the cells could
be duplicated by incubating LDL in the presence of transition metals in
the absence of cells. Thus, oxidative modification induced by cells
appeared to be a biologically plausible modification of LDL that could
account for foam cell formation and the initiation, or at least
acceleration, of the atherosclerotic process.
Between 1985 and 1989, 62 papers were published about "oxidized
LDL"; between 1992 and January 1997, 727 papers were published about
"oxidized LDL." This intense interest springs largely from the
increasing evidence that oxidative modification of LDL plays a
significant role in experimental atherosclerosis and thus may represent
a target for interventions to slow the progress of the disease (11,
12).
This review is limited to oxidative modification of LDL, but it should
be noted that other modified forms of LDL (e.g. aggregated LDL or LDL-containing immune complexes) can also induce foam cell formation and could contribute to atherogenesis (13-15).
To put the following discussion of oxidized LDL and its
pathobiological effects into a context, we begin with a brief summary of current views on the initiation of the atherosclerotic lesion. More
detailed discussions are available elsewhere (12, 16).
An increase in plasma LDL levels leads to an increase in the adherence
of circulating monocytes to arterial endothelial cells and at the same
time to an increased rate of entry of LDL into the intima, resulting in
a higher steady state concentration of LDL in the intima. There the LDL
can undergo oxidative modification catalyzed by any of the major cell
types found in arterial lesions, i.e. endothelial cells,
smooth muscle cells, or macrophages. Even minimally oxidized LDL
(MM-LDL) can increase adherence and penetration of monocytes, in part
by stimulating release of MCP-1 from endothelial cells (17). MM-LDL can
also stimulate release of MCSF, which can induce differentiation of the
monocyte into a cell with the phenotypic pattern of the tissue
macrophage, including an increase in expression of SRA (18). More fully
oxidized LDL (OxLDL) is itself directly chemotactic for monocytes, and
it is also, of course, one of the major ligands for SRA and other
receptors on the arterial macrophage that contribute to foam cell
formation. Soon after a lesion is initiated there is fragmentation of
the internal elastic membrane and migration of smooth muscle cells from
the media up into the intima. These smooth muscle cells do not normally
express SRA but can be induced to do so (19). This may be the basis for
the contribution that smooth muscle cells make to the foam cell
population. A centrally important point is that the fatty streak
lesion, while being clinically silent itself, is the precursor of the
more complex lesions that cause stenosis and limited blood flow. These
complex lesions ultimately represent the sites of thrombosis leading to
myocardial infarction.
Additional Potentially Proatherogenic Properties of Oxidized
LDL As already mentioned, the first property of oxidized LDL to be
discovered that makes it more atherogenic than native LDL is that it is
recognized by the scavenger receptors and can therefore give rise to
foam cells (7). Additional potentially proatherogenic properties became
apparent soon thereafter, including the fact that OxLDL is itself a
chemoattractant for monocytes (20) and that it inhibits the motility of
tissue macrophages (21). Oxidized LDL is cytotoxic for endothelial
cells in culture (22); it inhibits the vasodilatation that is normally
induced by NO (23); it is mitogenic for macrophages and smooth muscle
cells (24, 25); it can stimulate the release of MCP-1 and MCSF from
endothelial cells (17, 18). OxLDL is immunogenic, and autoantibodies
are commonly found both in animals and patients (26-28). Titers tend to be higher in patients with more rapidly progressive disease (27),
but, paradoxically, immunization of rabbits with OxLDL to raise
antibody titer actually inhibits lesion progression (29).
There are as many as 20 additional biological effects that have been
described, but almost none of these has been evaluated in
vivo. In any case, these examples will suffice to demonstrate that
the oxidative modification of LDL leads to a possibly very large array
of consequences above and beyond the generation of foam cells that
could be important in atherogenesis. It is important also to stress
that some of these biological effects can be exercised by minimally
modified LDL. Many of these appear to be attributable to partially
oxidized phospholipids that may mimic the effects of
platelet-activating factor or of autacoids (30, 31). The immunogenicity
of OxLDL appears also to be attributable in part to oxidized
phospholipids, possibly complexed with protein or other lipids (32,
33). Before leaving this topic, it is worth noting, as discussed
further below, that oxidation of the lipid-protein matrix of a plasma
membrane is somewhat analogous to the oxidation of an LDL particle.
Just as oxidized LDL can exert a number of effects, including
regulation of gene expression, oxidation of the plasma membrane of a
cell may give rise to analogous biological effects that could be
relevant during apoptosis or under conditions of high oxidative
stress.
Originally, oxidized LDL was defined primarily in terms of its
biological properties, notably the fact that it was no longer a ligand
for the native LDL receptor but was a ligand for the acetyl LDL
receptor and that its uptake by macrophages was therefore much more
rapid, sufficient to cause cholesterol accumulation. This degree of
oxidation could be effected by incubation overnight with cultured cells
in the appropriate medium or by incubation with 5-10 µM
Cu2+ for 8-16 h. Later studies showed that after much
gentler oxidative modification (too little to alter its binding by the
LDL receptor and yet not enough to make it a ligand for the acetyl LDL
receptor) the oxidized LDL had different and potentially very important biological properties of other kinds, including the ability to stimulate the release from endothelial cells of MCP-1 and MCSF (17,
18). This minimally oxidized LDL, designated "MM-LDL," is very
different from LDL incubated overnight with copper ions, which may
deserve to be redesignated as "maximally oxidized LDL." Obviously
there is potentially a continuous spectrum of degrees of oxidation and
a great deal of molecular heterogeneity in what we call "oxidized
LDL" (34). Even if oxidative conditions are controlled as precisely
as possible, the product will still vary from experiment to experiment
depending on the composition of the starting LDL. LDL particles rich in
polyunsaturated fatty acids are more readily oxidized than are LDL
particles enriched in saturated fatty acids or monounsaturated fatty
acids (35). The content of vitamin E and other naturally occurring
indigenous antioxidants will influence the susceptibility of LDL
preparations to oxidation under any given set of conditions. The
enormous complexity of the problem is evident when one considers that
the average LDL particle contains about 700 molecules of phospholipids,
600 of free cholesterol, 1600 of cholesterol esters, 185 of
triglycerides, and 1 of apolipoprotein B (apoB) containing 4536 amino
acid residues! Both the lipids and the protein are subject to oxidation
and both are indeed oxidized. Direct oxidative damage to proteins is
discussed by Berlett and Stadtman the previous Minireview in this
series (74), and almost certainly there is some direct apoB oxidation. Cholesterol is converted to oxysterols, especially at the 7-position. The polyunsaturated fatty acids in cholesterol esters, phospholipids, and triglycerides are subject to free radical-initiated oxidation and
can participate in chain reactions that amplify the extent of damage. A
key feature of LDL oxidation is the breakdown of these polyunsaturated
fatty acids to yield a broad array of smaller fragments, 3-9 carbons
in length, including aldehydes and ketones that can become conjugated
to other lipids (especially amino lipids) or to the apoB (36). For
example, malondialdehyde (or other aldehydes) generated during
oxidation can form Schiff bases with the As mentioned above, modification of LDL by endothelial cells
in vitro can be completely prevented by the addition of
antioxidants such as vitamin E or butylated hydroxytoluene (9). It is
almost completely inhibited also by the addition of as little as 5 or 10% fetal calf serum. How, then, can LDL undergo oxidative
modification in vivo? Even in the extracellular fluid one
would guess that the concentrations of antioxidants (proteins, vitamin
C, uric acid, etc.) would be ample to inhibit cell-induced oxidative
modification. Logic to the contrary notwithstanding, it does
get oxidized. 1) The lipoprotein fraction gently extracted from
atherosclerotic lesions (both rabbit and human) contains oxidized LDL,
identified both by its physical properties and by its recognition by
scavenger receptors (40); 2) immunohistochemistry using antibodies
generated against oxidized LDL demonstrates the presence of oxidized
LDL (or antigens very similar to it) in arterial lesions but not in normal artery (41); 3) both in animals and in humans autoantibodies that react with oxidized LDL have been demonstrated in the serum (41);
4) administration of antioxidants that can prevent oxidative modification of LDL slows the progression of atherosclerosis in several
experimental animal models, as discussed in more detail below.
How and where does this oxidation of LDL take place? That is really not
known, but it seems it must occur in sequestered microenvironments in
which the LDL is no longer protected by those antioxidant components that so effectively protect it in whole plasma or in extracellular fluid. When macrophages (and perhaps other cell types as well) adhere
to a substratum they behave a bit like the tentacles of an octopus,
i.e. microdomains of the cell membrane attach themselves to
the dish in a circular pattern creating microenvironments from which
large molecules are excluded (42). These "pockets" are so minute
that the composition of the fluid in them can be very rapidly changed
by virtue of transport across the cell membrane making up the "cap"
of the microenvironment. This might, for example, sharply reduce the
levels of antioxidants. If cells in vivo adhere to
connective tissue substrata (or to neighboring cells) in a similar
fashion, this postulated mechanism might explain oxidation of LDL
in vivo, but there is to date no experimental evidence one
way or the other.
Heavily acetylated LDL and heavily oxidized LDL injected intravenously
disappear from the plasma compartment with a very short half-life, only
a matter of minutes in the rat or in the rabbit. This largely reflects
extremely rapid uptake into hepatic Kuppfer cells and sinusoidal
endothelial cells (43). These cells express the acetyl LDL receptor and
probably other receptors for OxLDL and are highly efficient in sweeping
it out of the plasma. Consequently one would not expect to find heavily
oxidized LDL in the plasma at any significant concentration because it
would have to be generated at an implausibly high rate. On the other
hand, because MM-LDL is not a ligand for the scavenger receptors, it
would probably have a half-life not much different from that of native
LDL and could build up in the plasma compartment. Similarly, LDL in
which a small percentage of lysine Enzymes and Tissues Involved in LDL Oxidation in Vivo Studies in cell culture have identified a number of enzyme systems
that could in principle play a role in the oxidation of LDL. These
include NADPH oxidase, 15-lipoxygenase, myeloperoxidase, the
mitochondrial electron transport system, and others. Which of these
contribute to LDL oxidation in vivo and to what extent is
still uncertain, but analysis of products isolated from atherosclerotic lesions strongly supports the involvement of lipoxygenases (44, 45) and
of myeloperoxidase (46). Again cell culture studies identify many cell
types capable of oxidizing LDL including endothelial cells, smooth
muscle cells, monocytes, macrophages, fibroblasts, neutrophils, and
others. Since oxidized LDL is present in arterial lesions at
significant concentrations, it seems reasonable to assume that the
cells characteristic of those lesions, i.e. endothelial cells, macrophages, and smooth muscle cells, are involved in its oxidation. However, there is no convincing in vivo evidence
to implicate one or another of these as more important than the
others.
While oxidation of LDL in the artery wall has received the most
attention, it seems very likely that oxidation of LDL takes place at
many other sites, perhaps at all sites of inflammation. Because of
increased vascular permeability at sites of inflammation, the
concentration of LDL in the inflammatory fluid would be higher than it
is in normal extracellular fluid. Because of the infiltration by
neutrophils and monocyte/macrophages the conditions for LDL oxidation
at inflammatory sites would be propitious. However, LDL oxidation at
peripheral sites would not have the same significance as oxidation of
LDL in the artery wall unless the LDL oxidized at peripheral
sites reenters the bloodstream and is subsequently delivered to the
artery. If LDL in the periphery were to undergo limited oxidation
before reentering the blood it would have a prolonged half-life, as
discussed above, and it could then be taken up into developing arterial
lesions. Being already partially oxidized, this LDL might make an
unusually large contribution to the further progression of the lesion.
Immunochemical studies have provided evidence for the presence of
oxidized LDL (or at least of antigens closely related to it) at sites
of inflammation (47). The functional significance of this remains to be
explored.
Antioxidant Inhibition of Atherogenesis in Experimental
Models If oxidative modification of LDL plays a significant role in
atherogenesis, its inhibition by an appropriate antioxidant should slow
the progression of the disease. Indeed this has now been demonstrated
in several different animal models (the LDL receptor-deficient rabbit,
the cholesterol-fed New Zealand White rabbit, the cholesterol-fed hamster, the cholesterol-fed cynomolgus monkey, the LDL
receptor-deficient mouse, and the apoprotein E-deficient mouse) and
using one of several different antioxidants (probucol, butylated
hydroxytoluene, diphenylphenylenediamine, and vitamin E) (see Ref. 12
for review and specific citations). A total of 23 studies has been
reported of which 16 were strongly positive (more than 50% inhibition
of the rate of progression), 2 were borderline, and 5 negative. An important question to be asked is whether the antioxidants exerted their inhibitory effect on lesion progression only because of their
antioxidant properties or, possibly, because of additional biological
properties. This is the same kind of problem that arises with the use
of any inhibitor in biology. In fact the first antioxidant tested,
probucol, does indeed have additional biological properties that might
be relevant (48), including the ability to inhibit interleukin-1
release and to increase expression of cholesterol ester transfer
protein. However, the fact that two antioxidants as structurally
diverse as probucol and diphenylphenylenediamine share the ability to
inhibit atherogenesis suggests that the effect is attributable
primarily to their shared antioxidant properties. Further evidence that
the effect depends upon antioxidant activity comes from the rough
parallelism observed in some studies between the effectiveness of these
compounds in protecting circulating LDL from oxidation in an ex
vivo test system and their effectiveness in inhibiting
atherogenesis (49). At this time there is insufficient evidence,
however, to allow a confident prediction of the anti-atherosclerotic effectiveness of a compound from its antioxidant effectiveness ex
vivo. It appears that some rather high threshold of antioxidant effect must be reached before any anti-atherosclerotic
effect is evident (49, 50). Even a 4-fold prolongation of conjugated diene lag time (a commonly used measure of the resistance of LDL to
oxidation) may still be inadequate. Yet many clinical correlations are
being accepted as meaningful when the diene conjugation lag time is
increased as little as 30%!
Receptors for OxLDL and Their Evolutionary Raison d'Etre OxLDL is bound and internalized by at least two and possibly three
different macrophage receptors. Because these receptors tend to have a
much broader ligand specificity than previously studied receptors they
have been designated "scavenger receptors" or "multiligand"
receptors (51, 52). The best studied example is the original acetyl LDL
receptor, now redesignated scavenger receptor A, which occurs in two
differentially spliced forms (SRAI and SRAII) that have very similar
ligand binding specificity. Unlabeled acetyl LDL can only inhibit some
40-60% of the binding of OxLDL to mouse peritoneal macrophages (7,
8), and macrophages from mice in which SRA has been targeted show only
a partial defect in OxLDL binding, implying that additional receptors
are involved (53). Recent evidence shows that SRA can also play a role
in the adherence of macrophages to plastic surfaces (54) and to glycosylated collagen (55).
The pathogenetic role of SRA in atherogenesis has now been demonstrated
by crossing SRA-targeted mice with apoprotein E-targeted mice and
finding a highly significant 58% reduction in lesion severity (53).
Since OxLDL is one of the major naturally occurring ligands for SRA,
these findings further support the oxidative modification hypothesis of
atherogenesis.
The B class of scavenger receptors includes CD36 (56) and SR-B1 (57).
Finally, a receptor with scavenger receptor-like activity has been
cloned from Drosophila (58), and it has been designated as
the first member of a new class of scavenger receptors, SRC.
Recent studies have shown that macrosialin and its human homologue,
CD68, can bind OxLDL in ligand blots and that antibodies against CD68
can partially inhibit the binding and uptake of OxLDL by a human
monocyte-derived cell line, the THP-1 cell line (59, 60). However, only
a very small fraction of macrosialin or of CD68 is expressed on the
plasma membrane, and their importance in the uptake of OxLDL by normal
monocytes/macrophages remains to be further evaluated.
As mentioned above, receptors with at least some of the properties of
SRA and SRB can be found all the way back to Drosophila (58). Why have these receptors persisted in evolution? Surely it can
have nothing to do with any role they play in atherogenesis. Atherosclerosis is almost exclusively a human disease, and in any case,
its clinical effects occur after the procreational period is over so
there cannot be any selective genetic pressure (positive or negative).
We have suggested that oxidative damage to a cell membrane may generate
lipid-protein products similar to those found also in oxidatively
damaged LDL (61). Indeed, the binding of oxidatively damaged red blood
cells to macrophages is competitively inhibited by OxLDL but,
interestingly, not by acetyl LDL. The binding of apoptotic thymocytes
to macrophages is also inhibited by oxidized LDL (62), a finding
compatible with the hypothesis. The two best studied receptors for
oxidized LDL, SRA and CD36, also bind apoptotic cells. The role of CD36
in this respect has been extensively studied (63) and appears to
involve cooperative interaction with Is OxLDL Relevant to the Human Disease? The basic pathobiology of experimental atherosclerosis appears to
be very much the same as that of the human disease, suggesting that
antioxidants should work in humans. Furthermore, epidemiologic studies
have repeatedly shown a negative correlation between levels of dietary
intake or plasma levels of antioxidant vitamins, on the one hand, and
risk of coronary heart disease, on the other (65). On the other hand,
the time scale over which lesions develop in animal models is very
short (weeks or months) compared with the time scale over which human
lesions evolve (decades). Also, the degree of antioxidant protection we
can achieve in humans may be less than that achieved in animal
studies.
Several clinical trials utilizing Vitamin E, on the other hand, is very effective in protecting
circulating LDL against oxidation ex vivo (69, 71, 72). The
degree of protection is a function of the extent to which the vitamin E
content of the circulating LDL is increased, and doses of 400-800
international units daily seem to be required for maximal protection.
Only two clinical trials have been reported in which vitamin E
supplementation has been used. In one of these the dosage (50 IU daily)
was inadequate to protect circulating LDL, and the negative result is
therefore not relevant (66). The other study utilized 400-800 IU of
vitamin E daily in a placebo-controlled, double-blind trial in patients
with established coronary heart disease (73). Those randomized to
vitamin E showed 47% fewer nonfatal myocardial infarctions and
cardiovascular deaths (the primary end point) than the control group,
and the result was significant at the p = 0.001 level.
Additional trials are in progress. Decisions about the use of
antioxidants in human atherosclerosis should be deferred until
additional data become available.
-amino groups of lysine
residues and can go on to generate cross-links between lipid and
protein or among lipid molecules. 4-Hydroxynonenal and other
,
-unsaturated aldehydes can conjugate preferentially by Michael
addition (37). During the oxidation of LDL to a form recognized by SRA,
40-50% of the reactive lysine
-amino groups become masked (38,
39). This may be a sufficient explanation of the shift in receptor
specificity because treatment of LDL in vitro with acetic
anhydride also generates a form of LDL recognized by SRA when 60% or
more of the lysine amino groups are masked. This, then, explains what
was somewhat perplexing at first, namely why oxidized LDL and acetyl
LDL should have overlapping receptor-binding specificities. In part, at
least, it would appear to be because both involve masking of lysine
-amino groups with consequent changes in protein charge and
configuration.
-amino groups have been masked
(but not enough to make it a ligand for the scavenger receptors) might have a half-life even longer than that of native LDL and could, again,
build up.
V
3
and thrombospondin. Recent studies show that peritoneal macrophages
from mice in which SRA has been "knocked out" show a deficit in the
phagocytosis of apoptotic thymocytes (64). Thus it may be that as we
search for receptors that bind oxidatively damaged LDL we are at the
same time on the trail of receptors whose primary function is to
recognize damaged (apoptotic) cells.
-carotene, designed primarily to
test its possible efficacy in preventing cancer, have also recorded
cardiovascular events (66-68). All of them have been negative with
respect to effects on either cancer or cardiovascular disease.
Unfortunately, it was not recognized until recently that
-carotene
is actually relatively ineffective in protecting LDL (much less
effective than vitamin E). Carefully conducted trials in human subjects
show that supplementation even with very large doses of
-carotene
(doses sufficient to increase the
-carotene concentration in the LDL
fraction severalfold) fails to protect the circulating LDL against
oxidation ex vivo (69, 70).
-Carotene is an effective
quencher of singlet oxygen, but it is much less effective as a
chain-interrupting antioxidant. To the extent that protection of
circulating LDL is a rough index of efficacy, these
-carotene trials
should not be considered appropriate tests of the oxidative
modification hypothesis.