Canadian Journal of Cardiology 28 (2012) 631 641

Review

The Biological Role of Inflammation in Atherosclerosis

Brian W. Wong, PhD, Anna Meredith, BSc, David Lin, BMLSc, and
Bruce M. McManus, PhD, MD
UBC James Hogg Research Centre, Institute for Heart and Lung Health, St Pauls Hospital, University of British Columbia, Vancouver,
British Columbia, Canada

See editorial by Verma et al., pages 619-622 of this issue.

ABSTRACT

RSUM

The concept of the involvement of inflammation in the pathogenesis of

atherosclerosis has existed since the 1800s, stemming from sentinel
pathologic observations made by Rudolf Virchow, Karl Rokitansky, and
others. Our understanding of the complex role played by immune and
inflammatory mediators in the initiation and progression of atherosclerosis has evolved considerably in the intervening years, and today,
a dramatically evolved understanding of these processes has led to
advances in both diagnostic and prognostic approaches, as well as
novel treatment modalities targeting inflammatory and immune mediators. Therapeutic interventions working through multiple mechanisms involved in atheroma pathogenesis, such as statins, which both
lower lipids and alter the inflammatory milieu in the vessel wall, hold
promise for the future. In this brief review, we explore the biological
role of inflammation in atherosclerosis, with a focus on cellular involvement in both acute and chronic inflammation, and outline novel
biomarkers of inflammation and atherosclerosis with a particular focus on the potential application of these novel approaches in improving strategies for disease diagnosis and management.

Le concept de limplication de linflammation dans la pathogense de

lathrosclrose existe depuis les annes 1800 et provient des observations pathologiques sentinelles de Rudolf Virchow, Karl Rokitansky
et dautres. Notre comprhension du rle complexe jou par les mdiateurs immuns et inflammatoires dans lapparition et la progression
de lathrosclrose a considrablement volu depuis ces annes, et
aujourdhui, une comprhension notablement volue de ces processus a men aux avances des approches diagnostiques et des approches pronostiques, aussi bien quaux nouvelles modalits de traitements ciblant les mdiateurs inflammatoires et immuns. Les
interventions thrapeutiques contribuant aux multiples mcanismes
de la pathogense de lathrome, dont les statines qui abaissent les
lipides et modifient le milieu inflammatoire de la paroi vasculaire, sont
prometteuses pour le futur. Dans cette brve revue, nous explorons le
rle biologique de linflammation dans lathrosclrose, en nous concentrant sur limplication cellulaire de linflammation aigu et de
linflammation chronique, et dressons les grandes lignes des nouveaux biomarqueurs de linflammation et de lathrosclrose en
portant une attention particulire lapplication potentielle de ces
nouvelles approches lamlioration des stratgies de diagnostic et
de prise en charge de la maladie.

Atherosclerosis is a chronic vascular disease which involves the

progressive occlusion of blood vessels. There is evidence to
suggest that this process is initiated early in life, during postnatal development and maturation. Through a combination of
various genetic, environmental, and behavioural factors, the
resulting vessel occlusion may lead to the development of acute
coronary syndromes with myocardial infarction and sudden
death.
The concept of inflammation playing a role in the initiation
and progression of atherosclerosis is now mature, originating in

the 1800s from observations made by the great German pathologists and others.1,2
Inflammation represents the bodys primordial detection
and alarm system aimed at the containment and elimination of
foreign toxins and microbial pathogens. Chronic inflammation
has become recognized in recent years as a contributory factor
in the development of numerous chronic diseases including
cardiovascular disease (CVD), widely affecting the general
population. Atherosclerotic plaque development is one such
inflammation-driven condition. The host inflammatory response and resultant cellular and soluble mediator mobilization is critical in innate immune responses and crucial for
host defense against infections, and for minimizing or repairing tissue damage; however, in the face of unrelenting
persistent inflammation, the initiation, progression, and degenerative features of chronic diseases like native atheroscle-

Received for publication April 10, 2012. Accepted June 27, 2012.
Corresponding author: Dr Bruce M. McManus, Room 166, 1081 Burrard
St, Vancouver, British Columbia V6Z 1Y6, Canada. Tel.: 1-604-806-8586;
fax: 1-604-806-9274.
E-mail: bruce.mcmanus@hli.ubc.ca
See page 638 for disclosure information.

0828-282X/$ see front matter 2012 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.cjca.2012.06.023

632

rosis or the vasculopathy of autoimmune disorders like

rheumatoid arthritis (RA) may result. As a result of steady
research, it has become increasingly apparent in recent years
that inflammation is a central mechanism involved in the
entire life cycle of atherosclerosis.
Multiple levels of evidence, from experimental models and
histopathologic assessment of human tissues, to systemic biomarkers and epidemiologic or clinical associations, have furthered our realization that inflammation and atherosclerosis
are closely intertwined. The sources of data and information
that underpin our evolving knowledge of atherosclerosis and
inflammation are necessarily complex and of multiple origins, a
reflection of the intricate biological interplays in such a disease
process. Even with our long-held inferences of inflammatory
processes in the disease, new research directions continue to
push us forward. Given the multifactorial nature of atherosclerosis, the evidence needed to settle our understanding of the
condition, and to improve clinical diagnosis, management, and
prevention, is necessarily multifaceted.
This review explores the biological role of inflammation in
atherosclerosis, first by examining historic evidence supporting
the concept, and then by detailing the individual cell types
involved in acute and chronic inflammation with respect to the
vessel wall, paying particular attention to their cellular and
molecular biological roles in atherogenesis. The importance of
particular endogenous mediators and insights from animal
models of atherosclerosis will be highlighted, and finally the
emergence of novel biomarkers of inflammation and atherosclerosis will be elaborated, with a focus on the potential application of these novel insights in improving disease diagnosis
and management.
From Early Evidence to Early Initiation
The concept of inflammation was first introduced by the
Roman scholar, Celsus, with the now synonymous cardinal
signs of inflammation: rubor (redness/hyperemia), dolor
(pain), calor (warmth/heat), and tumour (swelling/edema).
This paradigm was complemented by observations of the
Greek-come-Roman, Galen, adding the term function laesa,
referring to a loss or disturbance of function. In essence, pathology or disease is a displacement from homeostasis, and it is
the role of inflammation to sense this displacement and restore
homeostasis. The purpose of this inflammatory process is the
resolution of injury, pathogens, or infections, through the initiation of an appropriate wound healing response. While inflammatory responses are normal and necessary, chronic inflammation is not defined as persistence of acute inflammation,
but rather by the presence of lymphocytes, macrophages, and
plasma cells within the injured tissue. Chronic inflammation
represents a deviation from a biologic or physiologic response
to an abnormal pathologic process.
Throughout the 19th and 20th centuries, physiologists and
pathologists provided key characterizations of the biologic
changes that occur in the vessel wall in atherosclerosis. Concomitant with endothelial injury, Virchow suggested that irritation of the intimal layer of the vessel wall results in degenerative and inflammatory changes that promote cell proliferation
within the vessel wall.1 Rokitansky, when describing the atheromatous plaque, stated that the deposit cannot be regarded as
a product (exudation) of an inflammation in the arteries [but

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Volume 28 2012

as] an endogenous product derived from the blood, and for the
most part from the fibrin of the arterial blood . . ..2 Thus, the
idea that inflammation may play a role in atherosclerosis stemming from circulating stimuli is one that has its roots in the
origins of pathology, and has continued to be refined in parallel
with this discipline for more than 150 years. With the clarity of
hindsight, it is exciting to revisit these fundamental observations, which have laid the foundation for modern day experiments that have augmented our understanding of the atherosclerotic process.
In 1958, Russell Holman and his team provided key observations in individuals from New Orleans, Guatemala, and
Costa Rica, defining fatty streaks as one of the earliest features
of atherosclerosis.3,4 Progression from fatty streaks to fibrous
plaques, leading to eventual hemorrhage, ulceration, or thrombosis, is believed to stem from fatty streaks. However, it was
noted that not every fatty streak progresses to arterial occlusive
disease.3,4 In this process, a slowly progressive inflammatory
and reparative reaction was noted in the surrounding tissues
(Fig. 1).4
If we pursue the notion of inflammation as a biological
process underlying atherosclerosis, one is compelled to consider the pathogenesis of atherosclerotic plaque as a series of
responses: to injury, to lipid retention, and so forth. In 1973,
work published by Russell Ross and John Glomset first described smooth muscle cell proliferation and migration as key
events in the formation of atherosclerotic lesions.5 Ross explored the role of endothelial denudation and macrophage ingress in atherogenesis,6,7 and further characterized the involvement of T cells and lymphocytes.8,9 He further went on to
provide a conceptual framework for factors which might be
responsible for the endothelial injury initiating atherosclerosis,
including blood flow patterns, lipids and lipoproteins, free radicals, systemic hypertension, plasma homocysteine, infection,
and other potential proinflammatory factors.10
The idea of atherosclerosis as a biologic response was
furthered by the work of Ira Tabas and Kevin Williams, who
championed the notion that atherogenesis may evolve primarily as a response to the retention of lipid and lipoprotein
retention within the vessel wall, emphasizing this event as
the fundamental cause and initiating event.11 Indeed, the
subendothelial retention of lipoproteins has not only been
clearly demonstrated, but also has been shown to be necessary in atherogenesis.12,13 Coupled with these initiating
events, intimal formation has been reported to appear during the perinatal period, when cell proliferation is apparently abundant.14 Insights from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) research
program shed further light on the initiation and progression
of atherosclerosis from qualitative and quantitative observations in young adults,15 where the presence of fatty streaks,
raised lesions, and early calcifications was characterized in computer template-assessed epicardial coronary arteries and similarly opened aortae.16
Overall, it is clear from this early work that not only the
initiating events, but also the biologic responses occur early and
progress throughout life. Central to the foregoing observations
regarding the evolution of the atherosclerotic plaque, these
changes are accompanied by inflammatory responses to the
vessel wall, specially shaped by the unique microenvironment
of the atherosclerotic plaque and its forerunners.

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633

Figure 1. Evolution of human atherosclerosis. Fatty streaks represent one of the earliest visible lesions in atherogenesis, and have been observed
early in life. These fatty streaks often evolve into fibrous plaques, coupled with intimal hyperplasia and accompanying changes to the extracellular
matrix. As these fibrous plaques further grow, they may progress toward several outcomes depending on microenvironmental and environmental
factors: [i] occlusive thrombosis, as a result of fibrous cap rupture because of the combination of endothelial cell loss revealing the prothrombotic
basal lamina, degradation of the fibrous cap as a result of proteases or proteinases or possibly through volume expansion and puncture as a
result of cholesterol crystals; [ii] intraplaque hemorrhage, as a result of abnormal neovascularization, vascular permeability, or increasing plaque
volume; [iii] stenosis, as a result of progressive luminal reduction by plaques leading to further lipid-richness, fibrosis, and hardening of the
arteries; or [iv] mural thrombic accretion, as a result of excess subendothelial accumulation of fibrin and platelets.

Inflammatory Subsets and Augmenting Factors

in Atherosclerosis
In the past 2 decades, characterization of the presence and
role of inflammatory mediators in atherogenesis and atherosclerosis has revealed the cellular effectors and the regulatory
cytokines, chemokines, growth factors, and humoural factors
at play.17-19
Inflammation in the vessel wall proceeds as cascades, which
begin with endothelial cell activation, resulting in the expression of adhesion molecules on the cell surface (intracellular
adhesion molecules), vascular cell adhesion molecules, selectins, integrins, and others, and the production and release of
proinflammatory cytokines and chemokines (tumour necrosis
factor [TNF]-), interferons (IFNs), monocyte chemoattractant protein-1, stromal cell-derived factor-1, macrophage inflammatory protein-1 , fractalkine, and others. These classic
responses may be augmented by other factors, including reactive oxygen species (ROS), hypoxia, and an altered growth
factor and cytokine milieu (Fig. 2).
Hypoxia is a strong stimulus for neovascularization, resulting in the formation of extensive vascular networks in vessel
walls. As the intimal layer expands, because of intimal smooth
muscle cell proliferation and matrix elaboration, the diffusion
limit for oxygen and nutrients creates a hypoxic niche. As well,
lipid insudation, retention, and modification occurring in
these regions foster the formation of a lipid-rich degenerative
core, which further serves to increase local hypoxia. This environment results in the stabilization of hypoxia-inducible factors (HIFs) through oxygen-sensitive mechanisms, resulting in
the activation of gene programs which promote survival in
conditions of limited oxygen. This milieu includes the preferential expression of glycolytic enzymes, allowing energy generation in an oxygen-independent fashion, and also proangiogenic gene programs, most importantly including vascular

endothelial growth factor (VEGF). VEGF promote angiogenesis extending into hypoxic regions in a biological attempt to
restore vascular perfusion. However, the microenvironmental
admix of growth factors and cytokines within the growing neointima may augment the proliferation and leakiness of these
neovessels. Edema occurs in inflammatory diseases when the
rate of plasma leakage from blood vessels exceeds the drainage
through lymphatic vessels and other exit routes. The extent to
which lymphatic growth occurs to compensate for increased
leakage during inflammation and atherosclerosis remains unclear. Nevertheless, the biological attempt to resolve hypoxia
within the growing atherosclerotic lesion is stimulated, adding
further to biologically abnormal responses.
The vasa vasora have been implicated in the development of
intimal hyperplasia.20 Vasa vasora may facilitate leukocyte entry into lesions, allow perfusion of the vessel wall beyond diffusion limits from the arterial lumen, or be the cause of intraplaque hemorrhage.21,22 Lack of lymphatic growth coupled
with neovascularization within the plaque may serve to further
the accumulation of immune mediators and cells in vessel
walls, suggesting that permeability-inducing factors such as
VEGF, a potent mediator of angiogenesis, may play an important role in this balance. Interestingly, VEGF-D is the only
family member not induced by hypoxia.23 It is likely that the
expression of VEGF family members, receptors, and cofactors
ultimately determines neovascularization and vascular permeability within the atherosclerotic plaque, and such may serve as
an interesting target for specific perturbation.
The T helper (Th)17 cell response has been shown to be
critical for exacerbating atherosclerosis in humans and mice,
serving a proinflammatory role in increasing inflammation and
promoting plaque instability.24,25 In addition, the balance of
Th17 and regulatory T cells has been demonstrated to be controlled by HIF-1.26 As well, hypoxia, HIF, and macrophages

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Volume 28 2012

Figure 2. Inflammatory mediators in atherosclerosis. (A) Numerous inflammatory cell types play major roles in mediating the inflammatory
response which is part of atherosclerosis, including T-cells, monocytes, and neutrophils. Early insudation and retention of lipid and lipoproteins
contribute to the initial and continued inflammatory response, especially when the lipids are oxidatively modified. These lipids and lipoproteins
are taken up by macrophages, dendritic cells, and smooth muscle cells to form lipid-laden foam cells. Proinflammatory cytokines, chemokines,
and growth factors serve to further elaborate this response in the vessel wall. Together, in addition to external factors such as hypoxia, reactive
oxygen species (ROS) and nitric oxide (NO) (in excess), inflammation within the atherosclerotic lesion fuels atheroprogression. (B) Early during
atherogenesis, monocytes and dendritic cells infiltrate into the vessel wall. (C) During progression of atherosclerosis, infiltrating monocytes
differentiate into macrophages and T-cells and other leukocytes enter the vessel wall. (D) As the atherosclerotic lesion further develops,
macrophage and foam cells predominate, and further serve to alter the plaque microenvironment, changing extracellular matrix composition and
decreasing smooth muscle cell content, predisposing to plaque rupture.

have been correlated with intraplaque angiogenesis in humans,27 and in apolipoprotein E-deficient mice, hypoxic macrophages have been shown to have increased sterol content as a
result of HIF-dependent impairment of cholesterol efflux and
induction of sterol synthesis.28 Macrophages exposed to hypoxia also secrete proteoglycans for which low-density lipoprotein (LDL) has higher affinity.29 In patients with severe coronary stenosis, the frequency of proline-to-serine single
nucleotide polymorphisms at the 582 residue of HIF-1 was
5-fold greater in patients without collateral growth,30 suggesting HIF-1 and hypoxia regulation may be a key genetic determinant of outcome in atherosclerosis. Taken together, factors such as hypoxia, ROS, or nitric oxide (in excess) in the
growing atheroma may also serve to further augment the ensuing inflammatory response and promote atherosclerosis.
Subendothelial monocyte recruitment has been demonstrated at sites predisposed to atherogenesis, and these cells may
not only differentiate into macrophages, but also dendritic
cells.31 Recently, the early uptake of lipids by resident intimal
dendritic cells has been shown to precede monocyte recruitment and differentiation, and contribute to the initiation of
atherosclerosis.32 Indeed, Paulson et al. demonstrated a 55%
reduction in early lipid accumulation in the aortic wall in the
absence of CD11c cells, suggesting that dendritic cells play a
key role in initial lipid uptake and retention in the vessel wall.32
Further, in hypercholesterolemia, dendritic cell migration to
lymph nodes is impaired.33 These reports highlight the importance of dendritic cells as mediators in atherogenesis and propagation of inflammation.

The role of the innate immune system in atherosclerosis and

CVD has been recently reviewed in detail,17,34 with growing
evidence suggesting that the toll-like receptor (TLR) pathways
are involved in plaque initiation and progression of atherosclerosis. The presence of TLRs within atherosclerotic plaques, and
on infiltrating leukocytes, provides evidence for the involvement of innate immunity in atherogenesis. TLR4, in particular, is expressed more highly in areas of plaque prone to rupture, and polymorphisms in this gene are associated with
susceptibility to coronary events and response to statin treatment in the setting of atherosclerotic complications.35 Oxidized LDL (oxLDL) exerts its proinflammatory role in part by
binding TLR4, and a knockout of the downstream effector of
TLR4 (MyD88) has been shown to have reduced atherosclerotic burden.36 Given the potential involvement of TLRs in the
progression of atherosclerosis, they have become significant
therapeutic targets. It has been demonstrated that statin treatment downregulates TLR4 expression in monocytes, and leads
to reduced expression of the downstream inflammatory mediators interleukin (IL)-6, IL-12, TNF-, and B7-1 ex vivo.37
Further work is needed to clarify the clinical potential of treatments targeting innate immunity in the setting of atherosclerosis. Phospholipase A2 (PLA2) is an enzyme frequently associated with lipoproteins and modifies phospholipids in these
particles to generate atherogenic species. Two members of the
PLA2 superfamily, liproprotein-associated PLA2 (Lp-PLA2)
and secretory PLA2 (sPLA2), have been linked to atherosclerosis. Lp-PLA2 is generated by inflammatory cells, including
monocytes, macrophages, and T-lymphocytes, is associated

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635

Figure 3. Progression of atherosclerosis and plaque formation. (A) The subendothelial retention of lipoproteins is an early initiating factor in
atherogenesis. (B) This initiating process results in monocyte chemotaxis in response to trapped low-density lipoprotein particles. As well, the
intimal layer expands, coupled with changes in extracellular matrix composition, including glycosaminoglycan-rich proteoglycans which have high
affinity for apolipoprotein B. (C) Lipoprotein uptake by macrophages results in foam cell formation, further advancing the atherosclerotic lesion.
Even minimally modified low-density lipoprotein can lead to cholesterol crystal formation, leading to Nod-like receptor P3 inflammasome priming
and macrophage activation. (D) Cholesterol crystals can be found not only within the lipid-rich necrotic core, but also in subendothelial areas both
intra- and extracellularly. Resultant macrophage and foam cell death may result directly because of cholesterol crystals, leading to release of
intracellular contents including lipoproteins, further expanding the necrotic core. Cholesterol crystals can also further activate Nod-like receptor
P3 inflammasomes, maintaining the pathological inflammatory response within the atherosclerotic lesion. SMC, smooth muscle cell.

with lipoproteins, particularly LDLs, and its expression has

been found to be increased in complex plaques. sPLA2 is an
acute phase reactant and is expressed by hepatocytes and
smooth muscle cells, and acts to modify phospholipids to generate proinflammatory lysophosphatidylcholine and oxidized
nonesterified fatty acids.38 Both Lp-PLA2 and sPLA2 modify
lipoproteins and lead to more highly oxidized LDL particles.38
OxLDL strongly induces inflammation by stimulating endothelial cells to release chemotactic proteins which result in increased recruitment of inflammatory leukocytes to the site of
the plaque.39 It has also been suggested that oxLDL promotes
the differentiation of monocytes into macrophages, thus contributing to the formation of foam cells. In addition to their
pathogenic role, levels of Lp-PLA2 and sPLA2 have been found
to be effective as risk markers for CVD and major adverse
cardiovascular events.40-43 In light of these data, development
of PLA2 inhibitors has become a promising area of research in
atherosclerosis.44,45 Inhibition of Lp-PLA2 has been shown to
reduce complex plaque development.46 However, because of
the large number of enzyme isoforms within this superfamily,
and the complexity of the PLA2 system, development of an
effective inhibitor may be challenging but warrants further investigation.

Another novel augmenting factor within the atherosclerotic

plaque is the cholesterol crystal. Cholesterol accumulates in the
vessel wall because of retention of lipoproteins that may result
in the formation of cholesterol crystals. Cholesterol crystal formation may be an initiating and exacerbating factor in atherosclerosis, as these crystals might directly induce cell injury and
apoptosis. For example, crystals might lead to damage of foam
cells, and within the lipid-rich necrotic cores characteristic of a
vulnerable plaque, the formation of cholesterol crystals may result
in volume expansions leading to plaque rupture (Fig. 3).47
The danger hypothesis pioneered by Polly Matzinger suggests that endogenous products from damaged cells and tissues,
termed damage-associated molecular patterns can initiate inflammatory responses in a manner similar to pathogens.48 Activation of pattern recognition receptors by alarm signals typically found within the cell, such as DNA, RNA, adenosine
triphosphate (ATP), uric crystals, hyaluron breakdown products, heat shock proteins, mitochondria, and others, lead to a
sequential cascade of differential regulatory events leading to
inflammation and a specific, localized, inflammatory response.49 Among other stimuli within the atherosclerotic milieu, cholesterol crystals may indeed act as stimuli, activating
inflammasomes to further inflammation. Plaque rupture lead-

636

ing to acute coronary syndromes can further be modulated

downstream of innate immunity.50,51 Macrophage activation
by damage-associated molecular patterns or oxidized lipids can
lead to foam cell formation or further activation of inflammatory signalling cascades. Particularly, the Th1 cytokine IFN-
inhibits collagen and extracellular matrix production by
smooth muscle cells,52 and can also inhibit smooth muscle cell
proliferation,53 together, altering plaque composition and stability. Elaboration of activated infiltrating immune cells can
lead to further extracellular matrix degradation.54
The inflammasome is a multiprotein complex, including
caspase-1, the apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain, a Nod-like receptor (NLR), and caspase 5. It is predominantly expressed in
myeloid cells and a central component of the innate immune
system. The exact composition of the inflammasome depends
on the specific activator, and can promote the activation of
inflammatory processes. Typically, foreign pathogens are recognized by pattern recognition receptors such as the cell surface
TLRs or C-type lectin receptors, or the intracellular NLRs or
retinoic acid-inducible gene 1 (RIG-I)-like helicase receptors.
The inflammasome activates an inflammatory cascade: the
inflammasome binds to p45 pro-caspase-1 molecules, mediating their cleavage into p20 and p10 subunits. The active form
of caspase-1 is then formed by 2 heterodimers, each containing
a p20 and p10 subunit.55 Activated caspase-1 can lead to a
number of disparate responses to the initial inflammatory insult, including the proteolytic cleavage of pro-ILs such as IL-1
and IL-18,56 inhibition of glycolytic enzymes,57 activation of
lipid biosynthesis,58 and the secretion of pro-IL-1, a mediator
of wound repair.59
IL-1 and IL-1 have been shown to promote inflammation in response to Western diets in mouse models of atherosclerosis.60 It has been suggested that cells within the vessel wall
mediate IL-1-dependent interactions with infiltrating myeloid
cells, and IL-1 activity furthers the inflammatory cascade
through the upregulation of cytokines, chemokines, and adhesion molecules on endothelial cells.61 IL-1 can also stimulate
smooth muscle cell and fibroblast proliferation,62 and taken
together, highlight the intimate role IL-1 signalling plays in
multiple steps in the initiation and progression of atherosclerosis. To this point, genetic inactivation of IL-1 signalling in
apolipoprotein E knockout mice displayed reduced atherosclerosis.63 As such, the effect of IL-1 inhibition on the prevention of recurrent cardiovascular effects is currently being investigated in the Canakinumab Anti-inflammatory Thrombosis
Outcomes Study (CANTOS) using a monoclonal antibody
directed against IL-1.64
The NLR family consists of NLRP1, NLRP3, and NLRC4.
These molecules contain a nucleotide-binding domain which
can bind ribonucleotide phosphates which facilitates self-oligomerization, and a C-terminus leucine-rich repeat, which functions in the recognition of other pattern recognition receptors or
ligands. NLRP activates caspase-1 via its pyrin domain to recruit
the adaptor protein, apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain, forming 1 oligomer per cell consisting of 7 NLRP3 molecules.65
It has been shown that NLRP3 inflammasomes are required
for atherogenesis, and activated by cholesterol crystals.66 Indeed, the activation of NLRP3 inflammasomes by cholesterol
crystals has been shown to be an important link between cho-

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lesterol metabolism and inflammation in macrophages.67 Interestingly, statins have been demonstrated to alter cholesterol
crystallization and dissolve cholesterol crystals in human arteries, leading to plaque stabilization.68 In addition to the activation of inflammation by classic mediators such as infection and
oxLDL, targeting cholesterol crystals may be another strategy
in reducing inflammation in atherosclerosis. It has been shown
that statins, ethanol, and aspirin are protective against acute coronary syndromes, have anti-inflammatory properties, and more recently, that they are effective solvents of cholesterol crystals.
Whether the clinical utility of these compounds relates to their
direct effects on cholesterol crystals, or rather on inflammation as
a whole, remains to be fully elucidated.
Inflammatory Conditions, Atherosclerosis, and
Therapeutic Agents
Over a period of 25 years, our laboratory has attempted to
further the understanding of inflammation and immunity in
blood vessel disease. Our work on cardiac allograft vasculopathy (CAV) has shed light on myriad ways in which the immune
system can contribute to native atherosclerotic lesion development. In many ways, CAV is an idiosyncratic and accelerated
form of atheromatous disease, and as such, we have gained
significant insights from this allogeneic environment about the
interplay between conventional risk factors and immunity. In
both severe CAV and atherosclerosis, the endothelium is severely dysfunctional and damaged, fostering inflammation, increased intimal thickening, and, eventually, the development
of proliferative fibrous plaques and degenerative foci. CAV has
been reviewed in detail previously in relation to atherosclerosis.69 Other comparator vasculopathies may also provide us
insights about native atherosclerosis. In the setting of systemic
lupus erythematosus (SLE) and RA, it has long been appreciated that CVD risk is higher than in the general population
with atherosclerosis and ischemic heart being common in both
patient populations; approximately 40% of RA mortality can
be attributed to CVD and accelerated atherosclerosis is an established complication of SLE.70
The human body contains 10 times as many microbial as
human cells, encoding 100 times as many genes as the human
genome,71 and their contribution to normal physiology and
pathology is increasingly being recognized. The National Institutes of Health-funded Human Microbiome Project and the
Canadian Institutes of Health Research Canadian Microbiome
Initiative are 2 programs currently under way to characterize
microorganisms found in the body and delineate their roles in
human biology. The gut harbors most of the microbes in the
body. Our appreciation of the role of gut microbia as regulators
of inflammation and atherosclerosis has evolved tremendously
in recent years,72 advanced in part by the metagenomic sequencing of the human gut microbial genome by Qin et al. in
2010.73 Exploration of the human gut microbiome has revealed the role gut flora play in nutrient availability and metabolism, and demonstrated the impact of these metabolic
characteristics on susceptibility to diabetes and insulin resistance.74,75 A recent study has demonstrated that abrogation of
gut flora through administration of antibiotics reduced atherosclerotic plaque development in mice by reducing dietary phosphatidyl choline metabolite-mediated plaque development.76
Further, modification of the commensal microbiome through

Wong et al.
Inflammation in Atherosclerosis

probiotic therapy could also reduce levels of atherogenic phospholipid levels, providing novel avenues of investigation in the
complex pathophysiologic interplay between host and microbiome in atherosclerosis.77 The role of commensal microbiota
in inflammatory and autoimmune diseases is an area of intense
investigation.78,79 Recent work has suggested that gut microbes can impact host innate immunity through activation of
TLR signalling. Mice deficient in TLR5 show signs of hyperlipidemia, hypertension, insulin resistance, and adiposity characteristic of metabolic syndrome, in parallel with altered gut
flora. This altered gut flora was sufficient to induce metabolic
syndrome when transferred to wild type mice, providing strong
evidence of the significant impact of gut flora on metabolic and
inflammatory dysregulation.80 Altered gut flora has also been
implicated in other inflammatory and autoimmune diseases
including inflammatory bowel disease, celiac disease, type 1
diabetes, and rheumatic diseases.79
The study of the atheroma in these autoimmune conditions
is complexa key challenge being the immunosuppressive
treatments, including corticosteroids, used in these diseases.
These drugs have associated toxicity and can also increase
blood lipids and modulate other traditional risk factors, in addition to certain immune responses, making the discrimination
of their specific contribution to atherosclerosis challenging. Yet
for sure, observed increases in both fatal and nonfatal CVD in
RA and SLE populations results from both the chronic inflammation characteristic of the diseases themselves and the manner
in which these immune and inflammatory processes interact
with traditional cardiovascular risk factors. The role of autoantibodies, inflammatory proteins, and cytokine profiles in these
patients is an area of intense study, and the dysregulation of
cytokines in these diseases represents a significant mechanism
linking autoimmunity and atheroma formation. Cytokine production by leukocytes, dendritic and natural killer cells, as well
as vascular smooth muscle cells and endothelial cells, contribute to systemic inflammation and consequent activation of
complement, production of C-reactive protein (CRP), and altered lipid profiles, all of which participate in atherogenesis.
Proatherogenic cytokines such as IL-6, IL-17, IFN-, TNF-,
and macrophage inhibitory factor have been identified in the
setting of inflammatory diseases that may provide targets of
treatment as well as novel diagnostic biomarker opportunities.
TNF antagonists, infliximab, adalimumab, and etanercept, are
used in RA and have been shown to control disease activity,
reduce systemic inflammation, and lower the incidence of
CVD events.81-83 Novel treatment approaches in the setting of
SLE using antibodies or small molecule inhibitors targeting
cytokines, their receptors, and related signalling mechanisms,
have been used with some success. For example, rituximab, an
anti-CD20 monoclonal antibody, has been shown to improve
the atheroprotective factors like high-density lipoprotein cholesterol and its related total cholesterol/high-density lipoprotein ratio, likely through a reduction in systemic inflammation,
although further studies are needed to determine if this effect
will translate into improved CVD rates in this patient population.84 It is important, however, to note that in essence all TNF
inhibitors are immunosuppressive, and the lessons learned
from CAV as a model of accelerated atherosclerosis indicate
that immunosuppression alone will likely not be enough to
stem progression and pathogenesis of atherosclerosis. The future of these anti-inflammatory treatments will ultimately need

637

to address the inherent redundancy in inflammatory pathways

and cytokine roles, and approach the interplay of these mediators through systems biology or network approaches in contrast to the current method of targeting mediators in isolation.
Statins remain the most widely used class of drugs in both
primary and secondary prevention of CVD. These drugs exhibit both a lipid-lowering action as well as immunomodulatory effects.85,86 The Pravastatin or Atorvastatin Evaluation
and Infection Therapy-Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22), Aggrastat to Zocor (A to Z),
and Reversal of Atherosclerosis With Agressive Lipid Lowering
(REVERSAL) post-hoc CRP studies revealed the anti-inflammatory effect of statins demonstrating the correlation between
atherosclerosis progression and circulation CRP, independent
of LDL cholesterol level.87-89 The Justification for the Use of
Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) trial later demonstrated similar
results, with rosuvastatin reducing incidence of major cardiovascular events in patients without hyperlipidemia presenting
with elevated CRP levels.90
Inflammatory Markers for Diagnosis and
Detection
The established role of inflammation in atherosclerosis has garnered interest in using inflammatory markers for diagnosis and
risk stratification. The clinical utility of inflammatory markers and
advanced lipoprotein testing was recently reviewed, highlighting
CRP, apolipoprotein B (ApoB) and lipoprotein (a).91
CRP binds to phosphocholine present on apoptotic cells,
oxLDL, and pneumococci, and may participate in the innate
immune response against phosphocholine-rich antigens. In
atherosclerosis, CRP can be generated within the plaque,92 and
CRP levels reflect the intensity of inflammation in atherosclerosis.93 ApoB has been shown to mediate the subendothelial
retention of lipoproteins early during atherogenesis, and ApoB
and lipoprotein (a) serve as surrogates for LDL cholesterol measurements. These markers vary in their utility and are not universally recommended for screening of patients, but can provide additional information for risk stratification or clinical
management in patients with a family history or for those at
risk of recurrent events. CRP was the only marker identified
which was recommended for evaluation in patients with intermediate risk of coronary heart disease on initial presentation.
CRP is a well-documented marker of inflammation and
acute phase protein, and thus potentially a significant player in
atherosclerosis, given the inflammatory component of this disease.83,84 It is expressed primarily by the liver in response to
elevated IL-6 and TNF-. Elevated CRP levels in the blood are
associated with increased CVD risk, and CRP is incorporated
into CVD risk assessment in the Reynolds Risk score. However, studies regarding the pathogenic role of CRP in CVD,
and whether its actions go beyond reflecting the inflammatory
milieu characteristic of the disease, remain inconclusive. CRP
has been found to colocalize with activated complement within
atheromatous plaques, and can induce adhesion molecule expression in human endothelial cells.94 In the JUPITER trial,
rosuvastatin was tested for prevention of vascular events in
17,802 healthy patients with elevated CRP but without hyperlipidemia, and was found to significantly reduced incidence of
major cardiovascular events (0.77 events per 100 person-years

638

in rosuvastatin group, 1.36 in placebo group). In this study,

treatment with rosuvastatin reduced CRP levels by 37% and
LDL levels by 50%.90 This trial indicates that reducing CRP
can improve outcomes even in patients without overt hyperlipidemia. It still remains unclear as to whether the benefit of
lowering CRP is because of a reduction of the inflammatory
state, actions of statin drugs, or the reduction in LDL levels.
Recent work has demonstrated that administration of human
CRP in diabetic rats increases the proinflammatory state in a
mechanism involving the activation of macrophages and upregulation of inflammatory mediators including protein kinase
C and nuclear factor kappa B.95 CRP has also been shown to
downregulate endothelial nitric oxide synthase production,96
which could further exacerbate endothelial dysfunction in the
setting of atherosclerotic plaque formation. However, a study
examining the effect of a CRP gene polymorphism resulting in
higher circulating levels did not find any association with cardiovascular events in the study population, casting doubt on
the causal role of CRP in CVD.97 Further work is needed to
dissect the role of CRP in atherosclerosis, beyond its utility as a
marker of risk.
A recent study, on the utility of coronary artery calcium
(CAC) and high-sensitivity CRP to predict all-cause and CVD
mortality mediated by atherosclerosis and systemic inflammation, found that while both measures improved coronary risk
prediction, this discriminatory action was mainly because of
CAC while high-sensitivity CRP played a greater role in individuals with very low CAC scores.98
Research focusing on the identification of novel biomarkers
for disease susceptibility and progression may allow us to preemptively treat specific subsets of individuals who are at risk of
disease, and identify those who will respond to current treatment modalities (ie, lipid-lowering/statins). At the present
time, although causal risk factors provide utility as therapeutic
targets, their use as predictive biomarkers of disease remains
limited and consequently patients can be misclassified into inappropriate risk levels.99 It is important to consider that susceptibility, and not only exposure, to causal factors remains a
significant contributor to overall patient risk of atherosclerosis.100 Genetic susceptibility studies will be valuable in deciphering these effects. A better understanding of individual susceptibility will provide an important adjunct to current risk
stratification tools and pave the road to further improvements
in clinical management and bring us closer to more personalized medicine.
Concluding Remarks
Atherosclerosis reflects processes of injury, immune response, inflammatory amplification, remodelling, reparation,
and potential restitution, but often degradation. Cumulative
evidence clearly establishes a role for inflammation in the vessel
wall. However, to date, clinical trials of antioxidant, anti-inflammatory, and antibacterial agents have shown no benefit in
reducing the burden of atherosclerotic disease.101 If indeed
inflammation is a reflection of the physiological responses to
resolving insults and/or injuries to the endothelium, further
investigation into strategies to remove these inflammatory
stimuli is warranted. The investigation of predisposing stimuli
such as shear stress, factors augmenting retention including
proteoglycans, lipoprotein lipase, sphingomyelinase, factors

Canadian Journal of Cardiology

Volume 28 2012

augmenting oxidation such as ROS, hypoxia, free radicals, or

nitric oxide, and the underlying autoimmunity and infection
which augment injury, still require more in depth study in
order to better understand the complex and ever-evolving relationship between inflammation and atherosclerosis.
Funding Sources
Canadian Institutes of Health Research, Heart & Stroke
Foundation of Canada, Genome Canada, Genome British Columbia, Networks of Centres of Excellence CECR Program.
Disclosures
The authors have no conflicts of interest to disclose.
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