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Review

The Biological Role of Inflammation in AtherosclerosisBrian W. Wong, PhD, Anna Meredith, BSc, David Lin, BMLSc, and

Bruce M. McManus, PhD, MDUBC 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.

ABSTRACTThe 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, andothers. Our understanding of the complex role played by immune and

inflammatory mediators in the initiation and progression of athero-

sclerosis 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 me-

diators. Therapeutic interventions working through multiple mecha-

nisms 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 in-

volvement in both acute and chronic inflammation, and outline novel

biomarkers of inflammation and atherosclerosis with a particular fo-

cus on the potential application of these novel approaches in improv-ing strategies for disease diagnosis and management.

RSUMLe concept de limplication de linflammation dans la pathogense de

lathrosclrose existe depuis les annes 1800 et provient des obser-

vations pathologiques sentinelles de Rudolf Virchow, Karl Rokitanskyet dautres. Notre comprhension du rle complexe jou par les m-

diateurs immuns et inflammatoires dans lapparition et la progression

de lathrosclrose a considrablement volu depuis ces annes, et

aujourdhui, une comprhension notablement volue de ces proces-

sus a men aux avances des approches diagnostiques et des ap-

proches pronostiques, aussi bien quaux nouvelles modalits de traite-

ments 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 con-

centrant sur limplication cellulaire de linflammation aigu et de

linflammation chronique, et dressons les grandes lignes des nou-

veaux biomarqueurs de linflammation et de lathrosclrose enportant 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 theprogressive occlusion of blood vessels. There is evidence tosuggest that this process is initiated early in life, during postna-tal development and maturation. Through a combination ofvarious genetic, environmental, and behavioural factors, the

resulting vessel occlusion may lead to the development of acutecoronary syndromes with myocardial infarction and suddendeath.

The concept of inflammation playing a role in the initiationand progression of atherosclerosis is now mature, originating in

the 1800s from observations made by the great German pa-thologists and others.1,2

Inflammation represents the bodys primordial detectionand alarm system aimed at the containment and elimination offoreign toxins and microbial pathogens. Chronic inflammation

has become recognized in recent years as a contributory factorin the development of numerous chronic diseases includingcardiovascular disease (CVD), widely affecting the generalpopulation. Atherosclerotic plaque development is one suchinflammation-driven condition. The host inflammatory re-sponse and resultant cellular and soluble mediator mobili-zation is critical in innate immune responses and crucial forhost defense against infections, and for minimizing or re-pairing tissue damage; however, in the face of unrelentingpersistent inflammation, the initiation, progression, and de-generative 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 BurrardSt, Vancouver, British Columbia V6Z 1Y6, Canada. Tel.:1-604-806-8586;fax:1-604-806-9274.

E-mail:[email protected] page 638 for disclosure information.

Canadian Journal of Cardiology 28 (2012) 631641

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
mailto:[email protected]://dx.doi.org/10.1016/j.cjca.2012.06.023http://dx.doi.org/10.1016/j.cjca.2012.06.023mailto:[email protected]

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rosis or the vasculopathy of autoimmune disorders likerheumatoid arthritis (RA) may result. As a result of steadyresearch, it has become increasingly apparent in recent yearsthat inflammation is a central mechanism involved in theentire life cycle of atherosclerosis.

Multiple levels of evidence, from experimental models andhistopathologic assessment of human tissues, to systemic bio-markers and epidemiologic or clinical associations, have fur-thered our realization that inflammation and atherosclerosisare closely intertwined. The sources of data and informationthat underpin our evolving knowledge of atherosclerosis andinflammation are necessarily complex and of multiple origins, areflection of the intricate biological interplays in such a diseaseprocess. Even with our long-held inferences of inflammatoryprocesses in the disease, new research directions continue topush us forward. Given the multifactorial nature of atheroscle-rosis, the evidence needed to settle our understanding of thecondition, and to improve clinical diagnosis, management, andprevention, is necessarily multifaceted.

This review explores the biological role of inflammation inatherosclerosis, first by examining historic evidence supporting

the concept, and then by detailing the individual cell typesinvolved in acute and chronic inflammation with respect to thevessel wall, paying particular attention to their cellular andmolecular biological roles in atherogenesis. The importance ofparticular endogenous mediators and insights from animalmodels of atherosclerosis will be highlighted, and finally theemergence of novel biomarkers of inflammation and athero-sclerosis will be elaborated, with a focus on the potential appli-cation of these novel insights in improving disease diagnosisand management.

From Early Evidence to Early Initiation

The concept of inflammation was first introduced by theRoman scholar, Celsus, with the now synonymous cardinalsigns of inflammation: rubor (redness/hyperemia), dolor(pain), calor (warmth/heat), and tumour (swelling/edema).This paradigm was complemented by observations of theGreek-come-Roman, Galen, adding the term function laesa,referring to a loss or disturbance of function. In essence, pa-thology or disease is a displacement from homeostasis, and it isthe role of inflammation to sense this displacement and restorehomeostasis. The purpose of this inflammatory process is theresolution of injury, pathogens, or infections, through the ini-tiation of an appropriate wound healing response. While in-flammatory responses are normal and necessary, chronic in-

flammation is not defined as persistence of acute inflammation,but rather by the presence of lymphocytes, macrophages, andplasma cells within the injured tissue. Chronic inflammationrepresents a deviation from a biologic or physiologic responseto an abnormal pathologic process.

Throughout the 19th and 20th centuries, physiologists andpathologists provided key characterizations of the biologicchanges that occur in the vessel wall in atherosclerosis. Con-comitant with endothelial injury, Virchow suggested that irri-tation of the intimal layer of the vessel wall results in degener-ative and inflammatorychanges that promote cell proliferationwithin the vessel wall.1 Rokitansky, when describing the ather-omatous plaque, stated that the deposit cannot be regarded asa product (exudation) of an inflammation in the arteries [but

as] an endogenous product derived from the blood, and for themost part from the fibrin of the arterial blood . . ..2 Thus, theidea that inflammation may play a role in atherosclerosis stem-ming from circulating stimuli is one that has its roots in theorigins of pathology, and has continued to be refined in parallelwith this discipline for more than 150 years. With the clarity ofhindsight, it is exciting to revisit these fundamental observa-tions, which have laid the foundation for modern day experi-ments that have augmented our understanding of the athero-sclerotic process.

In 1958, Russell Holman and his team provided key obser-vations in individuals from New Orleans, Guatemala, andCosta Rica, definingfatty streaks as one of the earliest featuresof atherosclerosis.3,4 Progression from fatty streaks to fibrousplaques, leading to eventual hemorrhage, ulceration, or throm-bosis, is believed to stem from fatty streaks. However, it wasnoted thatnot every fatty streak progresses to arterial occlusivedisease.3,4 In this process, a slowly progressive inflammatoryand 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 con-sider the pathogenesis of atherosclerotic plaque as a series ofresponses: to injury, to lipid retention, and so forth. In 1973,work published by Russell Ross and John Glomset first de-scribed smooth muscle cell proliferation and migration as keyevents in the formation of atherosclerotic lesions.5 Ross ex-plored the role of endothelial denudation and macrophage in-gress in atherogenesis,6,7 and further characterized the involve-ment of T cells and lymphocytes.8,9 He further went on toprovide a conceptual framework for factors which might beresponsible for the endothelial injury initiating atherosclerosis,including blood flow patterns, lipids and lipoproteins, free rad-icals, systemic hypertension, plasma homocysteine, infection,

and other potential proinflammatory factors.10

The idea of atherosclerosis as a biologic response wasfurthered by the work of Ira Tabas and Kevin Williams, whochampioned the notion that atherogenesis may evolve pri-marily as a response to the retention of lipid and lipoproteinretention within the vessel wall, emphasizing this event asthe fundamental cause and initiating event.11 Indeed, thesubendothelial retention of lipoproteins has not only beenclearly demonstrated,but also has been shown to be neces-sary in atherogenesis.12,13 Coupled with these initiatingevents, intimal formation has been reported to appear dur-ing the perinatal period, when cell proliferation is appar-ently abundant.14 Insights from the Pathobiological Deter-

minants of Atherosclerosis in Youth (PDAY) researchprogram shed further light on the initiation and progressionof atherosclerosis fromqualitative and quantitative observa-tions in young adults,15 where the presence of fatty streaks,raised lesions, and early calcifications was characterized in com-puter template-assessed epicardial coronary arteries and simi-larly opened aortae.16

Overall, it is clear from this early work that not only theinitiating events, but also the biologic responses occur early andprogress throughout life. Central to the foregoing observationsregarding the evolution of the atherosclerotic plaque, thesechanges are accompanied by inflammatory responses to thevessel wall, specially shaped by the unique microenvironmentof the atherosclerotic plaque and its forerunners.

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Inflammatory Subsets and Augmenting Factorsin Atherosclerosis

In the past 2 decades, characterization of the presence androle of inflammatory mediators in atherogenesis and athero-sclerosis has revealed the cellular effectors and the regulatorycytokines, chemokines, growth factors, and humoural factorsat play.17-19

Inflammation in the vessel wall proceeds as cascades, whichbegin with endothelial cell activation, resulting in the expres-sion of adhesion molecules on the cell surface (intracellularadhesion molecules), vascular cell adhesion molecules, selec-tins, integrins, and others, and the production and release ofproinflammatory cytokines and chemokines (tumour necrosisfactor [TNF]-), interferons (IFNs), monocyte chemoattrac-tant protein-1, stromal cell-derived factor-1, macrophage in-flammatory protein-1, fractalkine, and others. These classicresponses may be augmented by other factors, including reac-tive oxygen species (ROS), hypoxia, and an altered growthfactor and cytokine milieu (Fig. 2).

Hypoxia is a strong stimulus for neovascularization, result-

ing in the formation of extensive vascular networks in vesselwalls. As the intimal layer expands, because of intimal smoothmuscle cell proliferation and matrix elaboration, the diffusionlimit for oxygen and nutrients creates a hypoxic niche. As well,lipid insudation, retention, and modification occurring inthese regions foster the formation of a lipid-rich degenerativecore, which further serves to increase local hypoxia. This envi-ronment results in the stabilization of hypoxia-inducible fac-tors (HIFs) through oxygen-sensitive mechanisms, resulting inthe activation of gene programs which promote survival inconditions of limited oxygen. This milieu includes the prefer-ential expression of glycolytic enzymes, allowing energy gener-ation in an oxygen-independent fashion, and also proangio-genic gene programs, most importantly including vascular

endothelial growth factor (VEGF). VEGF promote angiogen-esis extending into hypoxic regions in a biological attempt torestore vascular perfusion. However, the microenvironmentaladmix of growth factors and cytokines within the growing neo-intima may augment the proliferation and leakiness of theseneovessels. Edema occurs in inflammatory diseases when therate of plasma leakage from blood vessels exceeds the drainage

through lymphatic vessels and other exit routes. The extent towhich lymphatic growth occurs to compensate for increasedleakage during inflammation and atherosclerosis remains un-clear. Nevertheless, the biological attempt to resolve hypoxiawithin the growing atherosclerotic lesion is stimulated, addingfurther to biologically abnormal responses.

The vasa vasora have been implicated in the development ofintimal hyperplasia.20 Vasa vasora may facilitate leukocyte en-try into lesions, allow perfusion of the vessel wall beyond dif-fusion limits from the arterial lumen, or be the cause of intra-plaque hemorrhage.21,22 Lack of lymphatic growth coupledwith neovascularization within the plaque may serve to furtherthe accumulation of immune mediators and cells in vessel

walls, suggesting that permeability-inducing factors such asVEGF, a potent mediator of angiogenesis, may play an impor-tant role in this balance. Interestingly, VEGF-D is the onlyfamily member not induced by hypoxia.23 It is likely that theexpression of VEGF family members, receptors, and cofactorsultimately determines neovascularization and vascular perme-ability within the atherosclerotic plaque, and such may serve asan interesting target for specific perturbation.

The T helper (Th)17 cell response has been shown to becritical for exacerbating atherosclerosis in humans and mice,serving a proinflammatory role i n increasing inflammation andpromoting plaque instability.24,25 In addition, the balance ofTh17 and regulatoryT cells has been demonstrated to be con-trolled by HIF-1.26As well, hypoxia, HIF, and macrophages

Figure 1.Evolution of human atherosclerosis. Fatty streaks represent one of the earliest visible lesions in atherogenesis, and have been observedearly 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 prothromboticbasal 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 plaquevolume;[iii] stenosis, as a result of progressive luminal reduction by plaques leading to further lipid-richness, fibrosis, and hardening of thearteries; or[iv] mural thrombic accretion, as a result of excess subendothelial accumulation of fibrin and platelets.

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have been correlated with intraplaque angiogenesis in hu-mans,27 and in apolipoprotein E-deficient mice, hypoxic mac-

rophages have been shown to have increased sterol content as aresult of HIF-dependent impairment of cholesterol efflux andinduction of sterol synthesis.28 Macrophages exposed to hyp-oxia also secrete proteoglycansfor which low-density lipopro-tein (LDL) has higher affinity.29 In patients with severe coro-nary stenosis, the frequency of proline-to-serine singlenucleotide polymorphisms at the 582 residue of HIF-1was5-fold greater in patients without collateral growth,30 suggest-ing HIF-1and hypoxia regulation may be a key genetic de-terminant of outcome in atherosclerosis. Taken together, fac-tors such as hypoxia, ROS, or nitric oxide (in excess) in thegrowing atheroma may also serve to further augment the ensu-ing inflammatory response and promote atherosclerosis.

Subendothelial monocyte recruitment has been demon-strated at sites predisposed to atherogenesis, and these cells maynot only differentiate into macrophages, but also dendriticcells.31 Recently, the early uptake of lipids by resident intimaldendritic cells has been shown to precede monocyte recruit-ment and differentiation, and contribute to the initiation ofatherosclerosis.32 Indeed, Paulson et al. demonstrated a 55%reduction in early lipid accumulation in the aortic wall in theabsence of CD11c cells, suggesting that dendritic cells play akey role in initial lipid uptake and retention in the vessel wall.32

Further, in hypercholesterolemia, dendritic cell migration tolymph nodes is impaired.33 These reports highlight the impor-tance of dendritic cells as mediators in atherogenesis and prop-agation of inflammation.

The role of the innate immune system in atherosclerosis andCVD has been recently reviewed in detail,17,34 with growing

evidence suggesting that the toll-like receptor (TLR) pathwaysare involved in plaque initiation and progression of atheroscle-rosis. The presence of TLRs within atherosclerotic plaques, andon infiltrating leukocytes, provides evidence for the involve-ment of innate immunity in atherogenesis. TLR4, in particu-lar, is expressed more highly in areas of plaque prone to rup-ture, and polymorphisms in this gene are associated withsusceptibility to coronary events and response to statintreat-ment in the setting of atherosclerotic complications.35 Oxi-dized LDL (oxLDL) exerts its proinflammatory role in part bybinding TLR4, and a knockout of the downstream effector ofTLR4 (MyD88) has been shown to have reduced atheroscle-rotic burden.36 Given thepotential involvement of TLRs in the

progression of atherosclerosis, they have become significanttherapeutic targets. It has been demonstrated that statin treat-ment downregulates TLR4 expression in monocytes, and leadsto reduced expression of the downstream inflammatory medi-ators interleukin (IL)-6, IL-12, TNF-, and B7-1 ex vivo.37

Further work is needed to clarify the clinical potential of treat-ments targeting innate immunity in the setting of atheroscle-rosis. Phospholipase A2 (PLA2) is an enzyme frequently asso-ciated with lipoproteins and modifies phospholipids in theseparticles to generate atherogenic species. Two members of thePLA2 superfamily, liproprotein-associated PLA2 (Lp-PLA2)and secretory PLA2 (sPLA2), have been linked to atheroscle-rosis. Lp-PLA2 is generated by inflammatory cells, includingmonocytes, macrophages, and T-lymphocytes, is associated

Figure 2. Inflammatory mediators in atherosclerosis. (A) Numerous inflammatory cell types play major roles in mediating the inflammatoryresponse 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 duringatherogenesis, monocytes and dendritic cells infiltrate into the vessel wall. (C) During progression of atherosclerosis, infiltrating monocytesdifferentiate 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.


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with lipoproteins, particularly LDLs, and its expression hasbeen found to be increased in complex plaques. sPLA2 is anacute phase reactant and is expressed by hepatocytes andsmooth muscle cells, and acts to modify phospholipids to gen-erate proinflammatory lysophosphatidylcholine and oxidizednonesterified fatty acids.38 Both Lp-PLA2 and sPLA2 modifylipoproteins and lead to more highly oxidized LDL particles.38

OxLDL strongly induces inflammation by stimulating endo-thelial cells to release chemotactic proteins which result in in-

creased recruitment of inflammatory leukocytes to the site ofthe plaque.39 It has also been suggested that oxLDL promotesthe differentiation of monocytes into macrophages, thus con-tributing to the formation of foam cells. In addition to theirpathogenic role, levels of Lp-PLA2 and sPLA2 have been foundto be effective as risk markers for CVD and major adversecardiovascular events.40-43 In light of these data, developmentof PLA2 inhibitorshas become a promising area of research inatherosclerosis.44,45 Inhibition of Lp-PLA2 has been shown toreduce complex plaque development.46 However, because ofthe large number of enzyme isoforms within this superfamily,and the complexity of the PLA2 system, development of aneffective inhibitor may be challenging but warrants further in-vestigation.

Another novel augmenting factor within the atheroscleroticplaque is the cholesterol crystal. Cholesterol accumulates in thevessel wall because of retention of lipoproteins that may resultin the formation of cholesterol crystals. Cholesterol crystal for-mation may be an initiating and exacerbating factor in athero-sclerosis, as these crystals might directly induce cell injury andapoptosis. For example, crystals might lead to damage of foamcells, and within the lipid-rich necrotic cores characteristic of avulnerable plaque, the formation of cholesterol crystals mayresult

in volume expansions leading to plaque rupture (Fig. 3).47

The danger hypothesis pioneered by Polly Matzinger sug-gests that endogenous products from damaged cells and tissues,termed damage-associated molecular patterns can initiatein-flammatory responses in a manner similar to pathogens.48Ac-tivation of pattern recognition receptors by alarm signals typi-cally found within the cell, such as DNA, RNA, adenosinetriphosphate (ATP), uric crystals, hyaluron breakdown prod-ucts, heat shock proteins, mitochondria, and others, lead to asequential cascade of differential regulatory events leading toinflammation and a specific, localized, inflammatory re-sponse.49 Among other stimuli within the atherosclerotic mi-lieu, cholesterol crystals may indeed act as stimuli, activatinginflammasomes to further inflammation. Plaque rupture lead-

Figure 3. Progression of atherosclerosis and plaque formation. (A) The subendothelial retention of lipoproteins is an early initiating factor inatherogenesis. (B) This initiating process results in monocyte chemotaxis in response to trapped low-density lipoprotein particles. As well, theintimal 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 bothintra- 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.


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ing to acute coronary syndromes canfurther be modulateddownstream of innate immunity.50,51 Macrophage activationby damage-associated molecular patterns or oxidized lipids canlead to foam cell formation or further activation of inflamma-tory signalling cascades. Particularly, the Th1 cytokine IFN-inhibits collagen and extracellular matrix production bysmooth musclecells,52 and can also inhibit smooth muscle cellproliferation,53 together, altering plaque composition and sta-bility. Elaboration of activated infiltrating immune cells canlead to further extracellular matrix degradation.54

The inflammasome is a multiprotein complex, includingcaspase-1, the apoptosis-associated speck-like protein contain-ing a C-terminal caspase-recruitment domain, a Nod-like re-ceptor (NLR), and caspase 5. It is predominantly expressed inmyeloid cells and a central component of the innate immunesystem. The exact composition of the inflammasome dependson the specific activator, and can promote the activation ofinflammatory processes. Typically, foreign pathogens are rec-ognized by pattern recognition receptors such as the cell surfaceTLRs or C-type lectin receptors, or the intracellular NLRs orretinoic acid-inducible gene 1 (RIG-I)-like helicase receptors.

The inflammasome activates an inflammatory cascade: theinflammasome binds to p45 pro-caspase-1 molecules, mediat-ing their cleavage into p20 and p10 subunits. The active formof caspase-1 is then formed by 2 heterodimers, each containinga p20 and p10 subunit.55 Activated caspase-1 can lead to anumber of disparate responses to the initial inflammatory in-sult, including the proteolytic cleavage of pro-ILs such as IL-1and IL-18,56 inhibition of glycolytic enzymes,57 activation oflipid biosynthesis,58 and the secretion of pro-IL-1, a mediatorof wound repair.59

IL-1and IL-1have been shown to promote inflamma-tion in response to Western diets in mouse models of athero-sclerosis.60 It has been suggested that cells within the vessel wall

mediate IL-1-dependent interactions with infiltrating myeloidcells, and IL-1 activity furthers the inflammatory cascadethrough the upregulation of cytokines,chemokines, and adhe-sion molecules on endothelial cells.61 IL-1 canalsostimulatesmooth muscle cell and fibroblast proliferation,62 and takentogether, highlight the intimate role IL-1 signalling plays inmultiple steps in the initiation and progression of atheroscle-rosis. To this point, genetic inactivation of IL-1 signalling inapolipoprotein E knockout mice displayed reduced atheroscle-rosis.63 As such, the effect of IL-1inhibition on the preven-tion of recurrent cardiovascular effects is currently being inves-tigated in the Canakinumab Anti-inflammatoryThrombosisOutcomes 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 whichcan bind ribonucleotide phosphates which facilitates self-oli-gomerization, and a C-terminus leucine-rich repeat, which func-tions in the recognition of other pattern recognition receptors orligands. NLRP activates caspase-1 via its pyrin domain to recruitthe adaptor protein, apoptosis-associated speck-like protein con-taining a C-terminal caspase-recruitment domain, forming 1 oli-gomer per cell consisting of 7 NLRP3 molecules.65

It has been shown that NLRP3 inflammasomes are requiredfor atherogenesis, and activated by cholesterol crystals.66 In-deed, the activation of NLRP3 inflammasomes by cholesterolcrystals has been shown to be an important link between cho-

lesterol metabolism and inflammation in macrophages.67 In-terestingly, statins have been demonstrated to alter cholesterolcrystallization and dissolve cholesterol crystals in human arter-ies, leading to plaque stabilization.68 In addition to the activa-tion of inflammation by classic mediators such as infection andoxLDL, targeting cholesterol crystals may be another strategyin reducing inflammation in atherosclerosis. It has been shownthat statins, ethanol, and aspirin are protective against acute coro-nary syndromes, have anti-inflammatory properties, and more re-cently, that they are effective solvents of cholesterol crystals.Whether the clinical utility of these compounds relates to theirdirect effects on cholesterol crystals, or rather on inflammation asa whole, remains to be fully elucidated.

Inflammatory Conditions, Atherosclerosis, andTherapeutic Agents

Over a period of 25 years, our laboratory has attempted tofurther the understanding of inflammation and immunity inblood vessel disease. Our work on cardiac allograft vasculopa-thy (CAV) has shed light on myriad ways in which the immunesystem can contribute to native atherosclerotic lesion develop-ment. In many ways, CAV is an idiosyncratic and acceleratedform of atheromatous disease, and as such, we have gainedsignificant insights from this allogeneic environment about theinterplay between conventional risk factors and immunity. Inboth severe CAV and atherosclerosis, the endothelium is se-verely dysfunctional and damaged, fostering inflammation, in-creased intimal thickening, and, eventually, the developmentof proliferative fibrous plaques and degenerative foci. CAV hasbeenreviewed in detail previously in relation to atherosclero-sis.69 Other comparator vasculopathies may also provide usinsights about native atherosclerosis. In the setting of systemiclupus erythematosus (SLE) and RA, it has long been appreci-

ated that CVD risk is higher than in the general populationwith atherosclerosis and ischemic heart being common in bothpatient populations; approximately 40% of RA mortality canbe attributed to CVD and accelerated atherosclerosis is an es-tablished complication of SLE.70

The human body contains 10 times as many microbial ashumancells, encoding 100 times as many genes as the humangenome,71 and their contribution to normal physiology andpathology is increasingly being recognized. The National In-stitutes of Health-funded Human Microbiome Project and theCanadian Institutes of Health Research Canadian MicrobiomeInitiative are 2 programs currently under way to characterizemicroorganisms found in the body and delineate their roles in

human biology. The gut harbors most of the microbes in thebody. Our appreciation of the role of gut microbia as regulatorsof inflammation and atherosclerosis has evolved tremendouslyin recent years,72 advanced in part by the metagenomic se-quencing of the human gut microbial genome by Qin et al. in2010.73 Exploration of the human gut microbiome has re-vealed the role gut flora play in nutrient availability and me-tabolism, and demonstrated the impact of these metaboliccharacteristics on susceptibility to diabetes and insulin resis-tance.74,75A recent study has demonstrated that abrogation ofgut flora through administration of antibiotics reduced athero-sclerotic plaque development in mice by reducing dietary phos-phatidyl choline metabolite-mediated plaque development.76

Further, modification of the commensal microbiome through


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probiotic therapy could also reduce levels of atherogenic phos-pholipid levels, providing novel avenues of investigation in thecomplex pathophysiologicinterplay between host and micro-biome in atherosclerosis.77 The role of commensal microbiotain inflammatory and autoimmune diseases is an area of intenseinvestigation.78,79 Recent work has suggested that gut mi-crobes can impact host innate immunity through activation ofTLR signalling. Mice deficient in TLR5 show signs of hyper-lipidemia, hypertension, insulin resistance, and adiposity char-acteristic of metabolic syndrome, in parallel with altered gutflora. This altered gut flora was sufficient to induce metabolicsyndrome when transferred to wild type mice, providing strongevidence of the significant impact of gut flora on metabolic andinflammatory dysregulation.80 Altered gut flora has also beenimplicated in other inflammatory and autoimmune diseasesincluding inflammatory bowel disease, celiac disease, type 1diabetes, and rheumatic diseases.79

The study of the atheroma in these autoimmune conditionsis complexa key challenge being the immunosuppressivetreatments, including corticosteroids, used in these diseases.These drugs have associated toxicity and can also increase

blood lipids and modulate other traditional risk factors, in ad-dition to certain immune responses, making the discriminationof their specific contribution to atherosclerosis challenging. Yetfor sure, observed increases in both fatal and nonfatal CVD inRA and SLE populations results from both the chronic inflam-mation characteristic of the diseases themselves and the mannerin which these immune and inflammatory processes interactwith traditional cardiovascular risk factors. The role of autoan-tibodies, inflammatory proteins, and cytokine profiles in thesepatients is an area of intense study, and the dysregulation ofcytokines in these diseases represents a significant mechanismlinking autoimmunity and atheroma formation. Cytokine pro-duction by leukocytes, dendritic and natural killer cells, as well

as vascular smooth muscle cells and endothelial cells, contrib-ute to systemic inflammation and consequent activation ofcomplement, production of C-reactive protein (CRP), and al-tered 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 thesetting of inflammatory diseases that may provide targets oftreatment as well as novel diagnostic biomarker opportunities.TNF antagonists, infliximab, adalimumab, and etanercept, areused in RA and have been shown to control disease activity,reduce systemic inflammation, and lower the incidence ofCVD events.81-83 Novel treatment approaches in the setting ofSLE using antibodies or small molecule inhibitors targeting

cytokines, their receptors, and related signalling mechanisms,have been used with some success. For example, rituximab, ananti-CD20 monoclonal antibody, has been shown to improvethe atheroprotective factors like high-density lipoprotein cho-lesterol and its related total cholesterol/high-density lipopro-tein ratio, likely through a reduction in systemic inflammation,although further studies are needed to determine if this effectwilltranslate into improved CVD rates in this patient popula-tion.84 It is important, however, to note that in essence all TNFinhibitors are immunosuppressive, and the lessons learnedfrom CAV as a model of accelerated atherosclerosis indicatethat immunosuppression alone will likely not be enough tostem progression and pathogenesis of atherosclerosis. The fu-ture of these anti-inflammatory treatments will ultimately need

to address the inherent redundancy in inflammatory pathwaysand cytokine roles, and approach the interplay of these medi-ators through systems biology or network approaches in con-trast to the current method of targeting mediators in isolation.Statins remain the most widely used class of drugs in bothprimary and secondary prevention of CVD. These drugs ex-hibit both alipid-lowering action as well as immunomodula-tory effects.85,86 The Pravastatin or Atorvastatin Evaluationand Infection Therapy-Thrombolysis in Myocardial Infarc-tion 22 (PROVE IT-TIMI 22), Aggrastat to Zocor (A to Z),andReversalofAtherosclerosis With AgressiveLipid Lowering(REVERSAL) post-hoc CRP studies revealed the anti-inflam-matory effect of statins demonstrating the correlation betweenatherosclerosis progression and circulation CRP, independentof LDL cholesterol level.87-89 TheJustification for the Use ofStatins in PrimaryPrevention: AnInterventionTrialEvaluat-ingRosuvastatin (JUPITER) trial later demonstrated similarresults, with rosuvastatin reducing incidence of major cardio-vascular events in patients without hyperlipidemia presentingwith elevated CRP levels.90

Inflammatory Markers for Diagnosis andDetection

The established roleof inflammationin atherosclerosishas gar-nered interest in using inflammatory markers for diagnosis andriskstratification. Theclinicalutilityof inflammatorymarkersandadvanced lipoprotein testing was recently reviewed, highlightingCRP, apolipoprotein B (ApoB) and lipoprotein (a).91

CRP binds to phosphocholine present on apoptotic cells,oxLDL, and pneumococci, and may participate in the innateimmune response against phosphocholine-rich antigens. Inatherosclerosis, CRP can be generated within the plaque,92 andCRPlevels reflect the intensity of inflammation in atheroscle-

rosis.93

ApoB has been shown to mediate the subendothelialretention of lipoproteins early during atherogenesis, and ApoBand lipoprotein (a) serve as surrogates for LDL cholesterol mea-surements. These markers vary in their utility and are not uni-versally recommended for screening of patients, but can pro-vide additional information for risk stratification or clinicalmanagement in patients with a family history or for those atrisk of recurrent events. CRP was the only marker identifiedwhich was recommended for evaluation in patients with inter-mediate risk of coronary heart disease on initial presentation.

CRP is a well-documented marker of inflammation andacute phase protein, and thus potentially a significant player inatherosclerosis, given the inflammatory component of this dis-

ease.83,84

It is expressed primarily by the liver in response toelevated IL-6 and TNF-. Elevated CRP levels in the blood areassociated with increased CVD risk, and CRP is incorporatedinto CVD risk assessment in the Reynolds Risk score. How-ever, studies regarding the pathogenic role of CRP in CVD,and whether its actions go beyond reflecting the inflammatorymilieu characteristic of the disease, remain inconclusive. CRPhas been found to colocalize with activated complement withinatheromatous plaques, and can induceadhesion molecule ex-pression in human endothelial cells.94 In the JUPITER trial,rosuvastatin was tested for prevention of vascular events in17,802 healthy patients with elevated CRP but without hyper-lipidemia, and was found to significantly reduced incidence ofmajor cardiovascular events (0.77 events per 100 person-years


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in rosuvastatin group, 1.36 in placebo group). In this study,treatment with rosuvastatin reduced CRP levels by 37% andLDL levels by 50%.90 This trial indicates that reducing CRPcan improve outcomes even in patients without overt hyperlip-idemia. It still remains unclear as to whether the benefit oflowering CRP is because of a reduction of the inflammatorystate, actions of statin drugs, or the reduction in LDL levels.Recent work has demonstrated that administration of humanCRP in diabetic rats increases the proinflammatory state in amechanism involving the activation of macrophages and up-regulation of inflammatory mediators including protein kinaseC and nuclear factor kappa B.95 CRP has also been showntodownregulate endothelial nitric oxide synthase production,96

which could further exacerbate endothelial dysfunction in thesetting of atherosclerotic plaque formation. However, a studyexamining the effect of a CRP gene polymorphism resulting inhigher circulating levels did not find any association with car-diovascular events in the studypopulation, casting doubt onthe causal role of CRP in CVD.97 Further work is needed todissect the role of CRP in atherosclerosis, beyond its utility as amarker of risk.

A recent study, on the utility of coronary artery calcium(CAC) and high-sensitivity CRP to predict all-cause and CVDmortality mediated by atherosclerosis and systemic inflamma-tion, found that while both measures improved coronary riskprediction, this discriminatory action was mainly because ofCAC while high-sensitivity CRPplayed a greater role in indi-viduals with very low CAC scores.98

Research focusing on the identification of novel biomarkersfor disease susceptibility and progression may allow us to pre-emptively treat specific subsets of individuals who are at risk ofdisease, and identify those who will respond to current treat-ment modalities (ie, lipid-lowering/statins). At the presenttime, although causal risk factors provide utility as therapeutic

targets, their use as predictive biomarkers of disease remainslimited and consequentlypatients can be misclassified into in-appropriate risk levels.99 It is important to consider that sus-ceptibility, and not only exposure, to causal factors remains asignificant contributor to overall patient risk of atherosclero-sis.100 Genetic susceptibility studies will be valuable in deci-phering these effects. A better understanding of individual sus-ceptibility will provide an important adjunct to current riskstratification tools and pave the road to further improvementsin clinical management and bring us closer to more personal-ized medicine.

Concluding RemarksAtherosclerosis reflects processes of injury, immune re-sponse, inflammatory amplification, remodelling, reparation,and potential restitution, but often degradation. Cumulativeevidence clearly establishes a role for inflammation in the vesselwall. However, to date, clinical trials of antioxidant, anti-in-flammatory, and antibacterial agents have shownnobenefit inreducing the burden of atherosclerotic disease.101 If indeedinflammation is a reflection of the physiological responses toresolving insults and/or injuries to the endothelium, furtherinvestigation into strategies to remove these inflammatorystimuli is warranted. The investigation of predisposing stimulisuch as shear stress, factors augmenting retention includingproteoglycans, lipoprotein lipase, sphingomyelinase, factors

augmenting oxidation such as ROS, hypoxia, free radicals, ornitric oxide, and the underlying autoimmunity and infectionwhich augment injury, still require more in depth study inorder to better understand the complex and ever-evolving re-lationship between inflammation and atherosclerosis.

Funding Sources

Canadian Institutes of Health Research, Heart & StrokeFoundation of Canada, Genome Canada, Genome British Co-lumbia, Networks of Centres of Excellence CECR Program.

DisclosuresThe authors have no conflicts of interest to disclose.

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