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Oxidized LDL antibodies in treatment and risk assessment of
atherosclerosis and associated cardiovascular disease
Jan Nilsson1, Gunilla Nordin Fredrikson1, Alexandru Schiopu1, Prediman K.
Shah2, Bo Jansson3 & Roland Carlsson3
1Department of Clinical Sciences, Malmö University Hospital, Lund University,
Sweden, 2Atherosclerosis Research Center, Cedars-Sinai Medical Center, UCLA
School of Medicine, Los Angeles, USA and 3Bioinvent International AB, Lund,
Sweden
Correspondence to:
Jan Nilsson
Department of Clinical Sciences
Entrance 33, 1st floor
Malmö University Hospital
S-205 02 Malmö, Sweden
Phone: 46 40 337684, Fax: 46 40 332550
Email: jan.nilsson@medforsk.mas.lu.se
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Abstract
Immune responses against oxidized forms of LDL play a critical role in
activation and regulation of the inflammatory process that characterizes all
stages of atherosclerosis. In humans oxidized LDL is targeted by both IgM and
IgG autoantibodies. Immunization of hypercholesterolemic animals with
oxidized LDL has been shown to inhibit atherosclerosis demonstrating that at
least some of these immune responses have a protective effect. The
identification of the structures in oxidized LDL that are responsible for
activation of immunity has made it possible to develop novel therapeutic
approaches for treatment of atherosclerosis based on active (vaccines) and
passive (antibodies) immunization. Studies performed in atherosclerosis-prone
mice demonstrate that both peptide-based vaccines and recombinant IgG
targeting epitopes in oxidized LDL significantly reduce atherosclerosis. There is
also evidence antibodies against oxidized LDL could also be used for imaging
atherosclerosis.
Key words: Atherosclerosis, low density lipoprotein, antibodies, immunity,
vaccine, inflammation, oxidation.
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Abbreviations
Apo B-100, Apolipoprotein B-100
CDR, Complementary determining regions
IMT, Carotid intima-media thickness
LDL, Low density lipoprotein
MDA, Malondialdehyde
NKT cells, Natural killer T cells
PAMP, Pathogen-associated molecular patterns
PAF, Platelet activating factor
PC, Phosphatidylcholine
scFv, single chain fragments
SR, Scavenger receptors
Th cell, T helper cell
TLR, Toll-like receptors
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Our understanding of the mechanisms involved in the development of
atherosclerosis has increased significantly over the last few decades and
irreversibly changed the way we view this silently progressive and potentially
deadly disease. Originally regarded as unavoidable consequence of continuous
lipid accumulation and age-related arterial degeneration the pioneering work of
Russell Ross and others in the early 70-ties culminating in the proposal of the
“response to injury” hypothesis of atherosclerosis focused attention on the
biological response of the artery wall and the role of smooth muscle cell
proliferation in particular [1]. Further studies came to identify lipid-induced
inflammation as the major culprit in induction of intimal fibrosis and
development of atherosclerotic plaques [2-5]. Additional interest in the role of
inflammation came from studies demonstrating that most acute cardiovascular
events are caused by an inflammation-driven degradation of plaque extracellular
matrix structures leading to plaque instability, surface erosion, rupture and
thrombus formation [6].
More recently it has become clear that the arterial inflammation associated with
development of atherosclerosis not only depends on accumulation of pro-
inflammatory and potentially cytotoxic lipids, but that it also is influenced by
complex innate and adaptive immune responses [7,8]. These findings are of
considerable clinical relevance because they suggest the immune system as a
possible target for development of novel therapeutical approaches for prevention
and treatment of cardiovascular disease. Interestingly, LDL is the one factor that
has remained in focus as the different concepts of atherogenesis have evolved
over the years. Most of the cholesterol that is deposited in the artery wall is
derived from LDL. Oxidation of LDL generates a number of potent pro-
inflammatory mediators and oxidized LDL is also a target for both innate and
adaptive immune responses. The importance of the latter mechanisms is
indicated by the observations that autoantibodies against oxidized LDL are
common in man [9] and that immunization of experimental animals with
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oxidized LDL leads to a partial protection against the development of
atherosclerosis [10,11]. In this review we will describe the role of immune
responses against oxidized LDL in atherosclerosis and discuss the possibilities
to use antibodies against epitopes in oxidized LDL in risk assessment,
prevention and treatment of atherosclerosis and cardiovascular events associated
with this disease.
Oxidized LDL and atherosclerosis
There is firm evidence linking cholesterol-transporting LDL particles to the
development of atherosclerosis. The LDL cholesterol level is the most important
risk factor for cardiovascular diseases in man, LDL cholesterol lowering drugs
(such as the statins) decrease the risk for development of cardiovascular events
by 30-40% and dietary as well as genetically induced hypercholesterolemia
activates development of atherosclerosis in experimental animals. The
mechanisms through which LDL contributes to atherogenesis have been
extensively studied and compelling experimental evidence has identified
oxidation of LDL as a key process in disease development [12]. LDL oxidation
is believed to take place primarily in the extracellular matrix of the artery wall
[13] where LDL particles aggregate bound to negatively charged proteoglycans
[14,15] (Figure 1). The mechanisms responsible for oxidation of LDL remain to
be fully understood but are believed to involve enzymes such as lipooxygenases.
Oxidation of LDL is associated with a number of structural modifications
including oxidative modification of phospholipid fatty acids, degradation of apo
B-100 into peptide fragments and modification of these fragments by aldehydes
derived from oxidized fatty acids [16]. All of these modifications are recognized
as neo-antigens by the immune system and play important roles in activation and
modulation of the subsequent inflammatory response [17].
Accumulation of oxidized LDL in the arterial wall stimulates local expression of
adhesion molecules and recruitment of monocytes and T cells [18,19].
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Infiltrating monocytes differentiate into scavenger receptor-expressing
macrophages that ingest the oxidized LDL, thus turning into cholesterol filled
foam cells if overloaded [2]. Intimal accumulations of such foam cells
constitute the first sign of a developing atherosclerotic plaque, the fatty streak.
A continuous inflow and oxidation of LDL increases the risk for establishment
of a chronic inflammatory state at affected arterial sites (typically at branching
points with low shear stress). Cytokines and growth factors released from
inflammatory cells then stimulate smooth muscle cells in the underlying media
to transform into fibroblast-like repair cells. These cells migrate into the intima
where they proliferate and secrete extracellular matrix proteins giving rise to a
fibromuscular atherosclerotic plaque. Plaques dominated by the fibro-
proliferative response are considered relatively benign as compared with plaques
in which lipid deposits, necrosis and inflammation prevail. In the latter type of
plaques macrophages secrete matrix metalloproteinases that degrade the fibrous
components increasing the risk for development of plaque instability, rupture
and thrombus formation [6]. Oxidized LDL contributes to this process both by
stimulating inflammation, the release of metalloproteinases and by being
cytotoxic for the smooth muscle cells that are responsible for replacing the
fibrous tissue degraded by macrophages [20]. Studies performed on human
plaques obtained at carotid surgery have shown that treatment with pravastatin
decreases the atherosclerotic plaques content of oxidized LDL and that this is
associated with decreased inflammation, improved smooth muscle cell viability
and increased collagen content [21]. However, statins are only able to prevent
30-40% of acute cardiovascular events demonstrating the need for development
of additional complementary therapeutical approaches.
Oxidized LDL interactions with the immune system
In general terms the immune system can be divided into two different
subsystems mediating innate and adaptive immune responses respectively.
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However, the two systems are tightly linked and co-regulate each other. Innate
immunity represents a fast but relatively blunt inflammatory and toxic response
to invading microorganisms, but does also interact with a number of modified
endogenous antigens. It utilises a number of receptors, cytokines, complement
factors, antibodies and other proteins encoded by germ-line genes. Adaptive
immunity is much more specific, but may take several days or even weeks to be
fully responsive. It involves a somatic rearrangement process in blast cells
leading to generation of a large number of T and B cells receptors and
immunoglobulins recognizing foreign antigens. Bridging these two branches of
the immune system are a number of cell types that have functional
characteristics of both responses including B1 cells, T cells and to some
extent also the natural killer T (NKT) cells [22]. Oxidized LDL is targeted by
both the innate and the adaptive immune system as well by the intermediate B-1
cells. The logical aim of these immune responses should be to remove the
potentially cytotoxic LDL particles from the vascular extracellular space before
they cause damage to surrounding cells. The mechanisms are similar to those
used by the immune system to remove apoptotic cells. This removal should
preferably be done without causing unnecessary harm to the affected tissue, i.e.
with as little inflammation as possible. In this respect the development of
atherosclerosis represents a failure of the immune system because the attempts
to remove oxidized LDL are accompanied by sustained inflammatory responses
and tissue damage. There are two aspects of this failure that appear equally
deleterious. First, the extra cellular lipid deposits characteristically present in
most atherosclerotic plaques suggests that the removal process has failed.
Second, the inflammation and scaring of the intima implies that the immune
system has been unable to perform the removal of oxidized LDL without
harming the tissue it is attempts to defend.
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An alternative possibility is that the inflammation and scaring occur only in
response to the toxic effect of oxidized lipids and that the immune system is a
passive by-stander in this process. However, as will be discussed below, studies
using mice with genetic deficiencies of various parts of the immune system
clearly shows that this system plays an active role in the development of
atherosclerosis. Why is then the immune system failing to protect the artery
wall? It may be that it has been adapted to clear the modest accumulations of
oxidized LDL likely to occur at low plasma cholesterol levels but that there has
been no evolutionary pressure to deal with the considerably higher cholesterol
levels occurring as a consequence of typical modern western diets. Another
complicating factor could be that certain parts of the immune system fail to
differentiate between epitopes in oxidized LDL and antigens present on the
surface of some micro-organisms due to molecular mimicry resulting in
activation of an unwarranted pro-inflammatory response.
Innate immune responses to oxidized LDL
Detection of so called pathogen-associated molecular patterns (PAMP) by
pattern recognition receptors on macrophages and dendritic cells is one of the
key elements of the innate immune system. There is a repertoire of pattern
recognition receptors binding a wide range of proteins, carbohydrates, lipids and
nucleic acids, but the ones considered most important in atherosclerosis are the
scavenger receptors (SR) and the Toll-like receptors (TLR) [23]. Scavenger
receptors mediate removal of modified lipoproteins, apoptotic cells and some
micro-organisms. TLRs are activated by LPS and other microbial antigens as
well as by some endogenous ligands (such as fibronectin extra-domain A and
heat shock protein 60) resulting in an induction of an inflammatory response.
These receptors are also expressed by endothelial cells and their activation is
likely to represent an important initial step in the atherosclerotic disease process.
There is compelling evidence from experimental studies that the arterial
inflammation that precedes plaque development is caused by accumulation of
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LDL in the extra-cellular matrix of the vessel. These LDL particles are modified
by a number of mechanisms including enzymatic degradation, aggregation and
oxidation resulting in generation of pro-inflammatory and potentially toxic lipid
peroxides, aldehydes and oxidized phospholipids [20]. The interactions between
these substances and endothelial cells result in an inflammatory response
activating infiltration of monocytes and T cells. Once resident in the arterial
intima monocytes will differentiate into macrophages expressing an array of
scavenger receptor including SR-A I and II, CD36, MARCO, SR-PSOX and
CD68 [24]. These will bind oxidized phospholipid epitopes on modified LDL,
mediating uptake and degradation of these particles. Excess cholesterol is stored
in intracellular lipid droplets leading to foam cell formation. Although this
appears to be functional mechanism for removal of a potentially toxic oxidized
LDL particle the observation that mice lacking SR-A [25] and CD36 [26]
develop less atherosclerosis suggest that this it is not without complications.
Uptake of modified LDL through scavenger receptors is in itself not coupled to
inflammatory activation. However, an innate inflammatory response may be
activated by phospholipid metabolites such as phosphatidylcholine (PC) and
platelet activating factor (PAF) released from LDL as a result of oxidation or by
toxic effects of oxidized LDL on vascular cells. Recent studies have also shown
that TLRs are expressed in human and murine atherosclerotic lesions and may
be induced by modified lipoproteins [27-30].
Natural antibodies against oxidized LDL
The natural antibodies can be seen as a humoral equivalent of the cellular
pattern recognition receptors. They are usually defined as antibodies that are
found in complete absence of exogenous antigenic stimulation. Natural
antibodies are not the result of gene rearrangement, but represent a family of
germline-encoded antibodies that are produced primarily by B-1 cells in the
spleen [31]. Most natural antibodies are IgM and poly-reactive. They provide a
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first line of defence against invading micro-organisms, but react also with self-
antigens associated with senescent cells and cellular debris. It is likely that these
“house keeping” functions are the reason why they have been so well preserved
during evaluation. It has been proposed that natural antibodies against modified
self antigens have an important role in protection against autoimmunity, but
their more precise function and mechanism of action remain largely unknown.
Interestingly, recent studies suggest that natural antibodies also constitute a key
component of the immune response against oxidized LDL. Palinski and co-
workers [32] established a panel of B cells hybridomas from apo E -/- mice.
These mice are known to have high levels of autoantibodies against oxidized
LDL and are prone to development of atherosclerosis. A number of clones were
selected that produced antibodies specifically binding to epitopes in oxidized
LDL. All clones were found to secrete IgM binding either to malondialdehyde
(MDA)-LDL or to copper-oxidized LDL. Subsequent studies showed that all
antibodies binding to copper-oxidized LDL recognized the same oxidized
phospholipid antigen, 1-palmitoyl-2 (5-oxovaleroyl) -sn-glycero-3
phosphorylcholine (POVPC) [33,34]. The antibodies recognized epitopes both
in the lipid moiety of oxidized LDL, as well as in de-lipidated, modified apo B-
100, suggesting that the antigen can exist as a free lipid as well as an adduct to
apo B-100. Epitopes recognized by these anti-phospholipid IgM were also
identified on the surface of apoptotic cells and antibody binding was shown to
inhibit scavenger receptor-mediated uptake of oxidized LDL and apoptotic cells
in macrophages [33,35]. Later studies revealed that the genes encoding the
antigen-binding site of these antibodies were identical to those encoding the T15
anti-phosphorylcholine antibodies produced by the B1 subset of B cells [36].
T15 antibodies also provide protection against several common infectious agents
including Streptococcus pneumoniae [37] Binder and co-workers [38] used this
information to study the functional role of anti-phospholipid antibodies in
atherosclerosis by immunizing LDL receptor knockout mice with Streptococcus
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pneumoniae. This treatment was found to result in induction of high levels of
oxidized LDL-specific IgM and a modest reduction of atherosclerosis.
Taken together these studies demonstrate that the presence of a natural antibody-
mediated immune response against a common or closely related phospholipids
epitopes present in oxidized LDL, apoptotic cells and Streptococcus
pneumoniae. They appear to have a protective effect against atherosclerosis
although their mechanism of action remains to be fully understood. Facilitation
of the removal of oxidized LDL, apoptotic cells and senescent cells could
potentially limit inflammation in atherosclerotic plaques. Inhibition of binding
and uptake of oxidized LDL by macrophage scavenger receptors represents
another possible anti-atherogenic effect of these antibodies. Notably, mice
deficient in the scavenger receptors CD36 [39] and SR-A [40] develop
significantly less atherosclerosis suggesting that the net effect of scavenger
receptor-mediated removal of oxidized LDL is proatherogenic. Finally, it is
possible that these natural antibodies protect against development of potentially
pro-inflammatory and atherogenic adaptive auto-immune responses against
oxidized LDL.
Adaptive immune responses against oxidized LDL
As described above oxidized LDL is taken up by scavenger receptors expressed
by macrophages and dendritic cells. The observations of oxidized LDL-specific
IgG in plasma and presence of oxidized LDL-specific T cells in atherosclerotic
plaques, as well as in the circulation, demonstrate that this uptake is associated
with activation of adaptive immune responses against oxidized LDL antigens.
The role of adaptive immunity in atherosclerosis has been extensively studied by
cross-breeding hypercholesterolemic, atherosclerosis-susceptible mice (such as
apo E -/- mice) with mice carrying genetic deletions of different components of
adaptive immunity [41,42]. Apo E -/- mice lacking functional T and B cells
develop significantly less atherosclerosis, whereas reconstitution of such mice
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with functional CD4+ T cells accelerates disease almost to the level of fully
immuno-competent apo E-/- mice [43]. In line with this, Zhou and co-workers
[44] recently reported decreased atherosclerosis in apo E -/- mice lacking CD4+ T
cells. The results of these studies generally points towards a pro-atherogenic role
of adaptive immunity in atherosclerosis. However, other studies indicate that the
role of adaptive immune responses is more complex. In order to characterize the
functional role of adaptive immune responses against oxidized LDL we [11] and
others [10] immunized hypercholesterolemic rabbits with oxidized LDL.
Unexpectedly, this was found to reduce atherosclerosis, an observation that was
subsequently confirmed in several studies using apo E -/- and LDL receptor -/-
mice [44-47].
How can these apparently contradictory findings be explained? One important
difference is that the experiments performed on immune-deficient mice did not
specifically study the role of immune responses against oxidized LDL, but rather
the net effect of all adaptive immune responses associated with the disease
process. Heat-shock protein has been identified as another important type of
autoantigen in atherosclerosis [48,49] (possible as a result of cross-reactivity
with bacterial heat shock protein) and these immune responses have been shown
to be strongly pro-atherogenic [50,51]. Accordingly, it is possible that disease
promoting immune responses against heat shock protein override the protective
effects of immune responses against oxidized LDL. An alternative explanation
may be that immunizations induced a shift in the immune response against
oxidized LDL from a pro-inflammatory Th1 towards an anti-
inflammatory/antibody-mediated Th2 response (Figure 1). Several lines of
evidence support the latter possibility. The pattern of cytokine expression in
atherosclerotic lesions is compatible with a pre-dominance of Th1 cells [52] and
animals deficient in the major Th1 cytokine, γ-interferon are relatively resistant
to development of atherosclerosis [53]. The immunization studies performed so
far have used Alum or Freunds’ complete followed by Freunds’ incomplete as
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adjuvants. Both Freunds’ incomplete adjuvant and Alum favour activation of
Th2 immune responses. A switch from naturally occurring Th1-specific
oxidized LDL IgG2a towards Th2-specific IgG1 has also been demonstrated in
several of these studies. Additional support for an important role of Th1/Th2
balance in atherosclerosis has come from studies demonstrating that LDL
receptor -/- mice lacking T-bet, the transcription factor that induces Th1
differentiation, have significantly less atherosclerosis [42]. Taken together these
data suggest that Th1 responses primarily contribute to the progression of
atherosclerosis while Th2 responses are protective. It remains to be fully
understood whether this protective effect is mediated by an enhanced antibody
production, the expression of anti-inflammatory cytokines (such as IL-10) or by
a combination of both. An alternative possibility that should be considered is
that immunization results in activation of regulatory T cells, a population of T
cells that down-regulate both Th1 and Th2 immune responses.
Characterization of peptide antigens in oxidized LDL
An important step towards understanding the role of adaptive immune responses
against oxidized LDL in atherosclerosis has been the characterization of the
antigens involved in this process. Activation of adaptive immunity typically
depends on T cell recognition of specific peptide sequences presented on MHC
class II molecules by macrophages and dendritic cells. The LDL particle
contains only one integrated protein, the 4500 amino acid apo B-100 that
mediates binding to the LDL receptor. During oxidation of LDL apo B-100
becomes degraded into multiple peptide fragments many of which form covalent
adducts with reactive aldehydes such as malondialdehyde (MDA) [54] (Figure
2). It is likely that these changes will result in formation of structures recognized
by the immune system as neo-antigens [17]. To investigate this possibility we
used a library of 302 20-aa-long polypeptides covering the complete apo B-100
sequence to construct a corresponding number of MDA- and non-MDA-
modified apo B-100 peptide ELISAs [55]. We then used these ELISAs to screen
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human plasma for detection of antibodies recognizing each particular sequence.
IgM against more than one hundred different MDA-modified apo B-100 peptide
sequences could be detected in plasma pooled from 50 healthy controls, whereas
detectable IgG levels only was found against a few sequences. It remains to be
elucidated how these IgM are related to the natural IgM antibodies that bind to
phospholipids in oxidized LDL, but our observations are compatible with the
existence of an innate-like, IgM-mediated immune response recognizing
multiple epitopes in modified apo B-100 along with an adaptive, IgG-mediated
response against a restricted number of sequences.
The functional role of adaptive immune responses against modified apo B-100
peptide sequences was studied in apo E -/- mice. Immunization with several of
the sequences found to induce adaptive immune responses in humans were
shown to significantly reduce the development of atherosclerosis in mice
[56,57]. The most effective atheroprotective response was found when
immunizations were made with the apo B-100 peptide sequences between amino
acids 16-35 (peptide 2), amino acids 631-650 (peptide 45) and amino acids
3136-3155 (peptide 210). The autoantibody levels in human plasma against
these 3 sequences usually differ markedly from each other, both IgM and IgG
against peptide 2 are non-detectable or very low, IgM levels against peptide 45
are high whereas IgG levels are low and both IgM and IgG levels against
peptide 210 are high. It will be important to determine the pathophysiological
relevance of these differences. It should also be kept in mind that the studies
performed so far demonstrating athero-protective effects of immunization with
oxidized LDL or oxidized LDL-specific antigens have been carried out in
relatively young animals while it remains to be established that this approach
also is effective also in more advanced stages of the disease.
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Therapeutic antibodies – a rapidly emerging new form of treatment
Utilisation of immune responses in experimental settings and eventually therapy
is an attractive approach. The immune system can be manipulated in two ways,
by active immunisation (vaccination) or by passive immunisation i.e. by
administration of pre-made antibodies. Vaccination has the potential to give
more long lasting effects whereas passive immunisation can give high, and
potentially protective levels of antibodies immediately. The latter effect could
potentially be of considerable benefit in treatment of vulnerable plaques and
unstable coronary disease. Utilization of the high specificity and selectivity of
antibodies in research and therapy was early recognized as an attractive
possibility. This possibility has now, with the advent of sophisticated technology
allowing generation of human antibodies reactive with human antigens, been
made a reality. Today, 17 antibodies have been registered as therapeutics and
many more are in clinical trials. In fact, antibodies represent the fastest growing
segment within the biotechnology industry and recently several therapeutic
antibody based drugs e.g. adalimumab (Humira) for treatment of rheumatoid
arthritis, rituximab (Rituxan) for treatment of non-Hodgkins lymphoma and
bevacizumab (Avastin) for treatment of colon carcinoma have entered the clinic
as registered products. The human or human like nature of these antibodies
important for therapeutic applications, in order not to induce immunological
responses against the administered protein based drug, was achieved by use of
technologies like chimerisation where mouse antibody specificities were
transferred to human constant antibody domains [58], or humanization where
the antigen binding parts the so called CDRs were transferred from specific
mouse antibodies into a pre-determined human antibody framework [59].
Finally completely human antibodies like Humira can be generated against, in
principle, any antigen including human antigens utilising recombinant library
technologies [60] or after immunization of mice made transgenic in the human
immunoglobulin locus [61].
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A recombinant library based technology called n-CoDeR [62] was utilised to
generate human antibodies against epitopes present on oxidised LDL. MDA-
modified peptides derived from human apo B-100 were used to select single
chain fragments (scFv) from the library. The resulting scFv, and human full
length IgG made utilising the scFv’s, were specific for the oxidised forms of the
peptides and also bound oxidised, but not to non-oxidised full length apo B-100
and LDL [63]. In addition, the antibodies demonstrated similar binding
specificities against mouse apo B-100 and mouse LDL and, thus, seemed
suitable for evaluation of therapeutic effects in mouse in vivo models.
Effect of antibody treatment on development of atherosclerosis
The effect of human recombinant anti-MDA- apo B-100 IgG on atherosclerosis
was studied in apo E -/- mice [63]. A limitation of this model is that the mice will
develop an immune response against human IgG that eventually will neutralize
the effect of the antibodies. Accordingly the treatment period was limited to 3
IgG injections one week apart starting when the mice had reached an age of 21
weeks. The extent of atherosclerosis was measured by en face Oil Red O
staining of the aorta at 25 weeks. Screening of a panel of antibodies against
MDA-peptide 45 and 210 suggested atheroprotective effects of IgG against both
peptide sequences and identified one antibody against MDA-peptide 45 (E3) as
the most effective. This antibody produced a 50% reduction in atherosclerosis
(Figure 3) and was also found to reduce inflammation and oxidized LDL
deposits in remaining plaques. We have subsequently developed new antibodies
against MDA-peptide 45 with higher binding affinity than the E3 antibody and
using several animal models shown that these antibodies may reduce
atherosclerosis to an even greater extent (Ström et al, unpublished data, Shiopu
et al, unpublished data). The underlying mechanism behind these effects is not
fully understood presently. However, it was noted that antibody treated animals
had less macrophages in their plaques than the untreated ones suggesting an
effect via inhibition of macrophage inflammatory activity by the antibodies [63].
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These findings have later been confirmed in in vitro and in vivo studies
demonstrating an inhibitory effect of release of pro-inflammatory cytokines
from macrophages by the antibodies (Frendéus et al. unpublished data).
Atherosclerotic plaque imaging using antibodies against oxidized LDL
The identification of vulnerable, rupture prone atherosclerosis plaques before
they give rise to acute cardiovascular events represents one of the major clinical
challenges in cardiology today. Routinely used techniques such as coronary
angiography and carotid ultrasound provide information regarding luminal
narrowing and plaque stenosis but have limited value in assessment of plaque
structure and risk for development of plaque rupture. The ability to identify high
cardiovascular subjects by determining the vascular calcium content using
electron-beam computer tomography is one example how analysis of plaque
constituents can provide information of clinical importance. However, since
calcium deposits do not necessarily distinguish rupture prone plaques from those
that are not rupture prone, the possibility to image vascular deposit of oxidized
LDL has the potential to be of even greater value in this respect. This possibility
has been explored by Tsimikas and co-workers [64] using an 125I-labeled mouse
monoclonal antibody specific for MDA-modified LDL (MDA2). Experiments
performed in hypercholesterolemic rabbits and LDL receptor -/- mice
demonstrate that intravenously injected 125I-MDA2 has a strong and specific
predilection for atherosclerotic lesions but not normal arterial tissue [65]. They
subsequently reported that stabilization of existing atherosclerotic lesions by
changing the high fat diet to normal chow resulted in a marked reduction of
plaque uptake of 125I-MDA2 [66]. These interesting studies suggest the
possibility of identifying vulnerable atherosclerotic plaques and monitoring
changes in plaque structure by imaging arterial uptake of radio-labelled oxidized
LDL antibodies in humans.
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Oxidized LDL autoantibodies as markers of atherosclerosis disease severity
and cardiovascular risk
The emerging evidence of an important role of immune responses against
oxidized LDL in atherosclerosis also suggest the possibility that the expression
of oxidized LDL autoantibodies could serve as a marker of disease and be used
to identify high risk patients. However, clinical studies of the association
between plasma levels of oxidized LDL autoantibodies and cardiovascular
disease have provided partly inconsistent results something that in part may be
due to difficulties in producing standardized antigens.
Some studies have reported increased levels of IgG against oxidized LDL in
patients with angiographically verified coronary artery disease [67-69] as
compared to healthy controls, while several others have failed to find any such
association [70-73]. An alternative approach to study the association between
oxidized LDL autoantibodies and atherosclerosis has been to use the carotid
intima-media thickness (IMT) as assessed by ultrasound. Karvonen et al [74]
found inverse associations between IgM and IgG autoantibody titers to oxidized
LDL and carotid IMT in middle-aged Finnish men and women. Associations
between low levels of IgG against oxidized LDL and increased carotid IMT has
also been reported in a study on Japanese subjects [75]. In contrast, a positive
association between oxidized LDL IgG and carotid IMT was observed in a study
on healthy middle-aged Swedish men [76].
There are still relatively few studies of the predictive value of autoantibodies in
cardiovascular disease and the existing data is primarily based on relatively
small case-control studies. Analyses of baseline samples in the Helsinki Heart
Study demonstrated elevated levels of IgG against oxidized LDL [77] in 130
subjects that developed acute myocardial infarction during the 5-year follow-up
period as compared to matched controls. In line with this Wu et al [78] found
that raised levels of antibodies against oxidized LDL at 50 years of age
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correlated positively with the incidence of myocardial infarction and mortality
related to myocardial infarction 10 to 20 years later.
The usefulness of oxidized LDL autoantibodies as markers of atherosclerotic
disease activity and as predictors of cardiovascular risk remains to be
established. The experience from other diseases involving organ-specific
autoimmune responses has convincingly demonstrated the potential of
autoantibodies as diagnostic tools, but also stresses the importance of careful
autoantigen characterization and antibody assay standardization. This may be
particularly important in the case of autoantibodies against LDL, since these
lipoprotein particles appear to contain a large number of different antigenic
structures. The recent progress in characterizing the molecular structure of the
neo-antigens formed in LDL as a result of oxidation may help to overcome this
problem. Using the apo B 100 peptide ELISAs described above we have been
able to demonstrate significant associations between IgM against several MDA-
apo B-100 peptide sequences and carotid artery IMT [55]. There were also
strong inverse associations between IgM levels and plasma oxidized LDL,
suggesting that these autoantibodies may function by removing oxidized LDL
from the circulation. IgM against three different MDA-modified peptide
sequences in apo B-100 were also shown to predict risk for development of
acute myocardial infarction and sudden cardiac death [55].
Taken together these epidemiological studies provide evidence for an
association between immune responses to oxidized LDL and the atherosclerotic
disease processes. However, they do not help to clarify the function of these
endogenous autoantibodies. One possibility is that association between high
autoantibody titers and disease severity could reflect a pro-atherogenic role of
the autoantibodies. Alternatively, their effect is protective and the extent of this
protective response parallels the severity of disease. As discussed above, the
available experimental evidence is in support of the latter alternative.
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Future developments – Immunotherapy targeting oxidised LDL for
treatment of atherosclerosis?
Oxidised LDL has been clearly implicated in the pathogenesis of atherosclerosis
and vaccination approaches as well as specific antibodies against oxidised
epitopes present on oxidised LDL have demonstrated atheroprotective effects in
in vivo animal models. As a target for therapeutic antibodies oxidised LDL has
many advantages. It is present at low concentrations only in the circulation
(about 0.1% of total LDL) where the consumption of administered antibodies or
antibodies produced after vaccination should be manageable and the risk for
potentially harmful complex formation low.
Using state of the art technologies vaccine formulations or pre-made fully
human antibodies, suitable as therapeutic drugs, and specific for the target can
be made and tested in relevant models. However, before this interesting
possibility can be tested in clinical trials a more thorough understanding of
mechanisms for the protective effects seen should have been established and a
category of patients that may benefit from such therapies identified.
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Figure legends
Figure 1. Immune responses against oxidized LDL. LDL particles entrapped
in the extracellular matrix of the artery wall become oxidized and activates an
inflammatory response leading to infiltration of monocytes and T cells. The
oxidized LDL is then taken up by macrophage scavenger receptors. While the
cholesterol is stored in lipid droplets other structures in oxidized LDL are
processed for presentation on CD1 (lipids) and MHC class II molecules
(peptides). In situations favouring maturation of naïve T cells into the Th1
phenotype this result in inflammation and disease progression. In contrast, if T
cells are stimulated to mature into Th2 or regulatory T cells inflammation and
disease progression appears to become inhibited.
Figure 2. Epitopes in oxidized LDL targeted by autoantibodies. IgM binds to
oxidized phospholipids and breakdown fragments of apo B-100, while IgG
binds only to apo B-100 fragments.
Figure 3. Effect of recombinant IgG against an aldehyde-modified apo B-
100 peptide sequence (amino acids 661-680) on atherosclerosis in apo E -/-
mice. *P<0.05 for difference between the IEI-E3 and FITC-8 IgG
22
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27
Figure 1.
28
Figure 2.
29
Figure 3.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.5 1 1.5 2 2.5
mg/dose
Oil Red O stained area in aortas (%)
FITC-8
IEI-E3
