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REVIEW TOPIC OF THE WEEK

Unraveling Vascular InflammationFrom Immunology to Imaging

Heather L. Teague, PHD,a Mark A. Ahlman, MD,b Abass Alavi, MD, PHD,c Denisa D. Wagner, PHD,d

Andrew H. Lichtman, MD, PHD,e Matthias Nahrendorf, MD, PHD,f Filip K. Swirski, PHD,f Frank Nestle, MD,g

Joel M. Gelfand, MD, MSCE,h Mariana J. Kaplan, MD,i Steven Grinspoon, MD,j Paul M. Ridker, MD,e

David E. Newby, DM, PHD,k Ahmed Tawakol, MD, PHD,l Zahi A. Fayad, PHD,m Nehal N. Mehta, MD, MSCEl

ABSTRACT

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Inflammation is a critical factor in early atherosclerosis and its progression to myocardial infarction. The search for

valid surrogate markers of arterial vascular inflammation led to the increasing use of positron emission tomography/

computed tomography. Indeed, vascular inflammation is associated with future risk for myocardial infarction and can

be modulated with short-term therapies, such as statins, that mitigate cardiovascular risk. However, to better

understand vascular inflammation and its mechanisms, a panel was recently convened of world experts in

immunology, human translational research, and positron emission tomographic vascular imaging. This contemporary

review first strives to understand the diverse roles of immune cells implicated in atherogenesis. Next, the authors

describe human chronic inflammatory disease models that can help elucidate the pathophysiology of vascular

inflammation. Finally, the authors review positron emission tomography–based imaging techniques to characterize

the vessel wall in vivo. (J Am Coll Cardiol 2017;70:1403–12) Published by Elsevier on behalf of the American College

of Cardiology Foundation.

m the aNational Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; bRadiology and Imaging

ences, Clinical Center, National Institutes of Health, Bethesda, Maryland; cUniversity of Pennsylvania, Philadelphia, Penn-

vania; dHarvard Medical School, Boston, Massachusetts; eBrigham and Women’s Hospital and Harvard Medical School, Boston,

ssachusetts; fCenter for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts;

ings College, London, United Kingdom; hPerelman School of Medicine, Philadelphia, Pennsylvania; iNational Institute of

thritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland; jProgram in Nutritional

tabolism, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; kBritish Heart Foundation

ntre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom; lCardiovascular Research Center,

ssachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; and the mIcahn School of Medicine at Mount

ai, New York, New York. Dr. Nestle is an employee of Sanofi. Dr. Gelfand has served as a consultant for Coherus (data and safety

nitoring board), Dermira, Janssen Biologics, Merck (data and safety monitoring board), Novartis, Regeneron, Dr. Reddy’s

boratories, Sanofi, and Pfizer; has received honoraria and research grants (to the Trustees of the University of Pennsylvania)

m Abbvie, Janssen, Novartis, Regeneron, Sanofi, Celgene, and Pfizer; has received payment for continuing medical education

rk related to psoriasis that was supported indirectly by Eli Lilly and Abbvie; and coholds a patent on resiquimod for the

atment of cutaneous T cell lymphoma. Dr. Grinspoon has served as a consultant for and received honoraria from Navidea

armaceuticals and Theratechnologies; and has received research grants (to the Massachusetts General Hospital) from Navidea

armaceuticals, KOWA Pharmaceuticals, Gilead Sciences, and Theratechnologies. Dr. Ridker has received investigator-initiated

earch support from the National Heart, Lung, and Blood Institute, Pfizer, AstraZenenca, Novartis, and Kowa; and is listed as a

nventor on patents held by the Brigham and Women’s Hospital that relate to the use of inflammatory biomarkers in cardio-

scular disease and diabetes that have been licensed to AstraZeneca and Siemens. Dr. Tawakol has served as a consultant for

telion Pharmaceuticals; and has received research grants (to the Massachusetts General Hospital) from Actelion and Genentech.

. Mehta is a full-time U.S. government employee; and has received research grants from Abbvie, Janssen, Novartis, and Celgene.

other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Teague and

lman contributed equally to this work. James Rudd, PhD, served as Guest Editor for this paper.

nuscript received June 16, 2017; revised manuscript received July 20, 2017, accepted July 20, 2017.

ABBR EV I A T I ON S

AND ACRONYMS

CT = computed tomographic

CV = cardiovascular

CVD = cardiovascular disease

FDG = 18F-fluorodeoxyglucose

HIV = human immunodeficiency

virus

IFN = interferon

MI = myocardial infarction

MR = magnetic resonance

NaF = 18F–sodium fluoride

NET = neutrophil extracellular

trap

PAD4 = peptidylarginine

deiminase–4

PET = positron emission

tomographic

SLE = systemic lupus

erythematosus

VI = vascular inflammation

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C ardiovascular disease (CVD) remainsthe leading cause of death world-wide, highlighting the need to eluci-

date its pathogenesis. Once considered apassive biological process, CVD is now recog-nized as an active, immune-driven processthat may begin in childhood (1). Currentresearch into the natural history of atheromadevelopment has implicated many immunecells, including phagocytes, lymphocytes,dendritic cells, and neutrophils (2). Becausethese cells play a major role in initiating pla-que development and complication, leuko-cytes are promising targets for acute andchronic atherosclerosis therapy. However,the complexity of the immune system andits role as a defensive force against infectionrequires novel tools to precisely identify andtreat only the inflammatory cells or processesthat promote atherosclerosis. Biomedicalengineering, specifically in human imaging,offers unique possibilities for diagnosing

and treating atherosclerotic plaque inflammationbefore cardiovascular (CV) events. Thus, interfacingnovel engineering to enhance human imaging withimmunology will be essential to accelerate advancesin management of this disease.

In this review, we begin with an overview of theemerging understanding of CVD as a systemicinflammatory disorder relating to monocytes, mac-rophages, neutrophils, and T cells. We then discussspecific human conditions with increased CVD risk tostudy the natural history of atherosclerosis, includinghuman immunodeficiency virus (HIV), systemic lupuserythematosus (SLE), and psoriasis as human modelsof vascular disease initiation and progression.Finally, we review current applications of positronemission tomographic (PET) imaging and emergingPET tracer agents used in vascular characterization.

IMMUNOLOGY OF INFLAMMATION AS IT

PERTAINS TO THE VESSEL WALL

MONOCYTES AND MACROPHAGES. The most numerouscells in atherosclerotic plaques are macrophages (3),leukocytes that are central to innate immunity. Inatherosclerosis, macrophage accumulation com-mences as bone marrow–derived, Ly6Chigh mono-cytes infiltrate the lesion. These Ly6Chighinflammatorymonocytes exit the bonemarrow in a C-Cmotif chemokine receptor 2–dependent manner,accumulate in the vessel wall, and differentiate intomacrophages, which are sustained through self-renewal (4). Notably, as atherosclerosis progresses,

local macrophage proliferation, rather than monocyterecruitment, becomes more important in lesiongrowth.

In addition to the accumulation of monocytes inatherosclerotic lesions, these innate immune cellscontribute to the biological response following amyocardial infarction (MI). Monocytes are bothdestructive and protective, in that they give rise toinfarct rupture and contribute to infarct healing.However, an overabundance of monocytes caninterfere with healing, resulting in heart failure. In anacute MI, an oversupply of monocytes to the aorta israpid, concomitant with a reduction in C-X-C motifchemokine ligand 12 expression in the bone marrow.Diminished C-X-C motif chemokine ligand 12 expres-sion enables myeloid cells and their progenitors to exitthe bone marrow and take up residence in the spleen,where they trigger extramedullary hematopoiesis (5).Additionally, differentiated leukocytes, especiallymonocytes and neutrophils, take up residence in end-organ tissues, giving rise to plaques and inflammation.The vascular sympathetic innervation (i.e., nervefibers traveling along the aorta and arterioles) plays arole in the increased emergency supply of leukocytes.In the periphery, sympathetic innervation activatesendothelial cells on the luminal surface of athero-sclerotic plaques, increasing adhesion moleculeexpression, which augments leukocyte recruitment(6). To directly investigate the role of the vascularsympathetic innervation system in atherogenesis, therole of stress in monocyte proliferation is a topic ofongoing investigation. Stress elevates noradrenalinelevels in the bone marrow and activates bone marrowstem cells (7). Hematopoietic stem and progenitor cellproliferation is significantly enhanced, C-X-C motifchemokine ligand 12 is lowered, and monocytes enterthe systemic circulation in increased numbers.Collectively, these mechanisms suggest a multiorgancommunication system that activates the bonemarrow through the sympathetic innervation system,increasing hematopoietic stem and progenitor cellproliferation and thus enhancing leukocytosis.Therefore, current studies aim to unravel the systemicmechanisms that control the production, recruitment,differentiation, and proliferation of monocytes andtheir descendent macrophages in atherosclerosis, todetermine how these processes can be balanced toexert themost benefit, and to elucidate specific controlpoints in atherogenesis.

NEUTROPHILS. Neutrophils are increasingly recog-nized in the initiation of atherosclerotic plaquedevelopment. Neutrophils are the initial immune cellto infiltrate inflammatory sites produced by injury or

FIGURE 1 Formation of Neutrophil Extracellular Traps Induces Vascular Damage

Neutrophil extracellular traps (NETs) in the timeline of deep vein thrombosis (DVT): a model. (A) DVT is initiated by local hypoxia and

activation of endothelial cells (ECs) as a result of flow restriction and disturbances. Activated endothelium releases ultralarge von Willebrand

factor (ULVWF) and P-selectin from Weibel-Palade bodies (WPBs), which mediate platelet and neutrophil adhesion. Activated platelets recruit

tissue factor (TF)–containing microparticles that enhance thrombin generation in the growing thrombus. (B) Activated platelets and

endothelium or other stimulus induce NET formation in adherent neutrophils. NETs provide an additional scaffold for platelet and red blood

cell (RBC) adhesion, promote fibrin formation, and exacerbate platelet and endothelial activation. (C) Plasmin, a disintegrin and

metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), and deoxyribonuclease (DNase) mediate thrombolysis by

degrading fibrin, ULVWF, and deoxyribonucleic acid, respectively. Monocytes/macrophages (Mø) release an additional source of DNase and

generate plasmin and promote restoration of blood flow (10).

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infection. Although the antimicrobial action of neu-trophils is indispensable to combat infection, theirmechanisms of action yield significant tissue damageand toxic debris. A newly identified defense mecha-nism is the ability of neutrophils to form neutrophilextracellular traps (NETs) (8); however, NET forma-tion, or “NETosis,” remains poorly understood.Currently, 2 distinct mechanisms have beendescribed, suicidal and vital NETosis. During suicidalNETosis, the internal membranes of the neutrophilsdissolve, followed by cell lysis and decondensedchromatin release (8). During an infectious process,neutrophils directly secrete nuclear contents from thecell without killing the neutrophils (9). Althoughmany questions remain unanswered regarding

NETosis, it is evident that NETs themselves areproinflammatory, induce endothelial and tissuedamage, and are highly prothrombotic (Figure 1) (10).Additionally, NETs provide a communication plat-form between neutrophils and macrophages, primingmacrophages to produce pro–interleukin-1b, culmi-nating in atherosclerotic plaque destabilization (11).

NET formation is a peptidylarginine deiminase–4(PAD4)–dependent mechanism induced by extracel-lular stimuli via microbes, activated platelets, cyto-kines, antibodies to neutrophils, hypoxia, andcholesterol crystals. Upon activation, PAD4 convertsarginine residues on histones to citrulline, triggeringchromatin decondensation and NET release (12).Consequently, PAD4 is expelled in conjunction with

FIGURE 2 Vascular Images of Chronically Inflamed Human Models

Representative 18F-fluorodeoxyglucose positron emission tomographic/computed tomographic imaging of the aorta in a healthy volunteer

(A), compared with the aortas of patients with human immunodeficiency virus (B), psoriasis (C), and systemic lupus erythematosus (D).

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a milieu of toxic proteins, resulting in citrullinationof surrounding proteins, altering their functionalproperties and producing neoantigens, which mayplay a role in autoimmune diseases (13). In a murinemodel of MI, ischemia of the heart induces NETosis.Furthermore, PAD4�/�, a murine model incapable ofNETosis, displays smaller infarct size and hassignificantly better heart function, demonstrated byan elevated ejection fraction subsequent to anischemic event (14). In aged or diabetic mice, neu-trophils are primed for NETosis, producing excessivethrombosis and inflammation (15). Myocardial NETdeposition delays wound healing, leading to fibrosisin the cardiac pressure-overload model. Intriguingly,spontaneous fibrosis of organs produced by aging isgreatly reduced in PAD4�/� mice. Functionally,PAD4�/� hearts are comparable with young murinehearts, and their systolic and diastolic function doesnot decline with age (15). Thus, there is recentinterest in further study of the specific roles ofneutrophils, NETosis, and the PAD4 pathway inatherosclerosis.

T CELLS. When immune effector mechanisms areactive within the blood vessel wall or endothelialsurface, vascular dysfunction, thrombosis, andischemia usually result. T cells play important roles inthe promotion and regulation of inflammationin atherosclerotic lesions. Human arteries haveinterferon (IFN)–g–producing and interleukin-17–producing CD4þ T cells in atherosclerotic lesions, andmouse studies have shown that IFN-g-producing Thelper 1 cells promote atheroma development (16).Consistent with the finding that T cells are involvedin plaque inflammation, murine models show that theB7-CD28 T-cell costimulatory pathway is involved inpromoting proatherogenic T-cell responses, as well asatheroprotective regulatory T-cell responses (17).Importantly, murine models indicate that inhibitorymembers of the B7-CD28 family, in particular PD-L1(B7-H1) expressed on antigen-presenting cells, endo-thelium, and the cells of various tissues, and itsreceptor on T cells, PD-1, are important in suppressingT cell–driven inflammation in arteriosclerosis andmyocytes (18). For example, mice lacking PD-L1 or

FIGURE 3 Fluorine-18–Sodium Fluoride Uptake in the

Coronary Arteries

A discrete focus of 18F–sodium fluoride uptake overlying an

otherwise heavily calcified left anterior descending coronary

artery, suggesting a locus of active calcification with potentially

increased vulnerability for rupture (arrow).

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PD-1 display a marked increase in CD4þ- and CD8þ-mediated inflammation in arterial lesions. Mice lack-ing PD-1 or PD-L1, or treated with PD-1 blockade, aremore susceptible to T cell–mediated myocardialinjury. Furthermore, IFN-g-induced up-regulation ofPD-L1 by heart endothelial cells in vitro or in vivoprotects against CD8þ cytotoxic T lymphocyte–mediated damage (19).

Mouse studies demonstrating the protective rolesof the PD-1 pathway in arteries and heart highlight thepossibility of increasing CV risks by targeting PD-1 orPD-L1 in cancer immunotherapy. In fact, many casesof acute severe lymphocytic myocarditis are nowbeing reported in the context of checkpoint blockadecancer immunotherapy, including patients treatedwith anti-PD-1 (20). Histopathologic analyses oftissues from some of these cases reveal up-regulationof endothelial human leukocyte antigen–antigen Drelated, as well as myocyte and endothelial PD-L1associated with T-cell infiltrates, consistent with anIFN-mediated effect.

Notably, CVD initiation and progression involvebiological activity from multiple immune cells, bothinnate and adaptive. Future studies deciphering theinterplay among these immune cells in CVD are crit-ical for developing therapies targeting CVD initiationin order to reduce clinical CVD outcomes andultimately decrease CVD prevalence.

HUMAN DISEASE MODELS OF IMAGING TO

STUDY VASCULAR INFLAMMATION

Currently, 2 large ongoing CV trials in patients withprior MI are testing if treatment of inflammation willreduce a second CV event: CIRT (CardiovascularInflammation Reduction Trial) and inhibition ofinterleukin-1B in CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study). There isan initial report that CANTOS met the primaryendpoint for a reduction in recurrent major adverse CVevents. These trials will provide critical data onwhether inhibition of nonspecific T-cell inflammation(methotrexate) or inflammasome activation (canaki-numab) reduces further CV events in high-risk pa-tients. As the results of these trials become available,emerging data from human chronic diseases associ-ated with high CV risk and systemic inflammationprovide models to understand vascular disease initia-tion and progression. Indeed, 18F-fluorodeoxyglucose(FDG) PET/computed tomographic (CT) imaging hasbeen used to characterize vascular inflammation (VI)in HIV infection, psoriasis, and SLE (Figure 2).HUMAN IMMUNODEFICIENCY VIRUS. HIV treatmenthas become very effective over the past decade;however, the rate of MI, stroke, and sudden cardiacdeath remains elevated 50% to 100% in HIV infection(21). Although dyslipidemia is more prevalent in HIVinfection, traditional risks, including dyslipidemia,hypertension, and diabetes, only account for 25% ofthe excess risk. Using coronary CT angiography, astudy demonstrated a high prevalence of noncalcifiedplaque with high-risk morphological features, asso-ciated with increased immune activation indexes (22)and inflammation, on FDG PET imaging thatremained elevated after effective antiretroviraltherapy (23).

Among patients with HIV infection, coronary pla-ques are often inflamed and noncalcified (24), andpatients exhibit increased myocardial fibrosis imagedby magnetic resonance (MR) (25). Imaging with FDGhas demonstrated that HIV-infected patients with anundetectable viral load have increased aortic target-to-background ratios compared with healthy controlsubjects and subjects with known CVD, indicatingsignificant arterial inflammation, even in the contextof immune restoration and viral suppression(Figure 2) (26). Together, coronary CT angiographyand FDG PET imaging have helped identify theunique pathophysiology of arterial inflammation andplaque in HIV infection, proved to be a readout for theefficacy of anti-inflammatory strategies now beingtargeted in HIV infection, including newer statins.Statins effectively decrease noncalcified plaque as

TABLE 1 A Summary of Agents and Their Potential Mechanisms of Uptake Applicable to

Vascular Inflammation Imaging

Agent (Ref. #) Potential Mechanism of Uptake

18F-FLT (62) Structural analogue of thymidine, images DNA synthesis withinatheroma

11C-PK11195 (63) Affinity for translocator protein, upregulated on inflammatorycells

18F-A85380 (64) Binds arterial nicotinic acetylcholine receptors, possibly relatedto vascular damage

18F-choline (65) Images increased cell wall synthesis within atheroma68Ga-DOTA-octreotate (60) Affinity for somatostatin receptors, which are highly expressed

on macrophages64Cu-ATSM (66) Trapped within cells in hypoxic state18F-MISO (67) Trapped within cells in hypoxic state68Ga-NOTA-RGD (68) Images neoangiogenesis as a result of hypoxia or chronic

inflammation64Cu-DOTA-CANF (69) Images neoangiogenesis via natriuretic peptide receptor

affinity18F-FDG A glucose analogue imaging increased metabolic rate in the

presence of inflammation and hypoxia18F–sodium fluoride (59) Images active calcification as a result of necrosis or

inflammation68Ga-CXCR4 (70) Images CXCR4 receptor expressed by inflammatory cells18F-florbetapen (61) Imaging b-amyloid plaque as a component of inflammation

11C-PK11195 ¼ 11C-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; A85380 ¼3-([2S]-azetidinylmethoxy)pyridine dihydrochloride; ATSM ¼ diacetyl-bis(N-methylthiosemicarbazone;CXCR4 ¼ C-X-C chemokine receptor type 4; DOTA-CANF ¼ 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid atrial natriuretic factor; FDG ¼ fluorodeoxyglucose; FLT ¼ fluorothymidine; MISO ¼ fluoromisonidazole;NOTA-RGD ¼ 1,4,7-triazacyclononane-N,N0 ,N00-triacetic acid arginine-glycine-aspartate.

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well as low-density lipoprotein in patients with HIVinfection (27). On the basis of these data, a large trialof 6,500 patients with HIV infection and a low tomoderate risk for CVD is now under way to testwhether pitavastatin can prevent CV events byreducing low-density lipoprotein and concomitantlyimprove inflammatory pathways of immune activa-tion (REPRIEVE [Randomized Trial to PreventVascular Events in HIV]; NCT02344290).

PSORIASIS. Psoriasis is a chronic T helper 1 cellT helper 17 cell inflammatory disease that affectsmore than 125 million people worldwide and about2% to 4% of the adult population, with the mostcommon type manifesting as plaque psoriasis.The pathophysiology of the disease is localizedskin inflammation, epidermal hyperproliferation,up-regulated T-cell and neutrophil activation, andincreases in C-reactive protein, serum amyloid A, andintercellular adhesion molecule 1 (28). Consistentwith chronically inflamed pathologies, largepopulation-based epidemiological studies suggestthat patients with psoriasis, particularly moderate tosevere, have an increased risk for MI, stroke, andCV mortality independent of traditional risk factors(29–31). Notably, studies demonstrate strong associ-ations of psoriasis and CVD with increasing severityof skin disease. Patients with moderate to severe

psoriasis experience premature mortality, dyingapproximately 5 years younger than their non-psoriatic counterparts in adjusted analysis (32). Over10-year periods, patients with moderate to severepsoriasis have about 6% excess risk for developing amajor CV event, beyond the conventional Framing-ham risk score calculation (33). Interestingly, trans-lational studies have demonstrated dramaticup-regulation of genes known to adversely affect CVrisk in the skin lesions of psoriasis. Furthermore,transgenic mouse models of psoriasis in which thegenetic construct is confined to the skin demonstrateaortic inflammation and thrombosis (34). Similarly,patients with psoriasis have an increased amount ofaortic FDG PET activity that is positively correlatedwith skin disease severity (35).

A series of randomized, placebo-controlled clinicaltrials are evaluating the impact of treatmentssuch as adalimumab compared with phototherapy(NCT01553058), ustekinumab (NCT02187172), andsecukinumab (NCT02690701) on aortic inflammation(measured by FDG PET imaging) and CV biomarkersin psoriasis. These trials will provide greater insightinto the ability of anti-inflammatory therapies todiminish the risk for CVD.SYSTEMIC LUPUS ERYTHEMATOSUS. Patients withSLE have a heightened risk for developing athero-sclerosis, including MI and stroke (36), with youngwomen having up to a 50-fold increased risk fordeveloping vascular events (37). Furthermore, evenpatients with SLE with no prior CVD events havesubclinical vascular dysfunction, endothelialdysfunction, arterial stiffness, myocardial perfusionabnormalities, carotid plaque, and coronary calcifi-cation not fully explained by Framingham risk scoreor by medications used to treat SLE (36).

Altered innate immune responses characteristic ofSLE contribute to the development of lupus vascul-opathy and atherosclerotic plaque formation (38).Type I IFNs, a group of cytokines elevated in manypatients with SLE, particularly during periods of flare,have pleiotropic deleterious roles in the vasculature(39). Type I IFNs independently associated withendothelial function, carotid plaque, and severity ofcoronary calcification in patients without histories ofCVD (40). Specifically, these cytokines promote animbalance of enhanced endothelial cell damage anddecreased vascular repair that may promote initiationof vasculopathy and contribute to the development offoam-cell formation and platelet activation. SLE isalso characterized by the presence of a distinct subsetof pathogenic neutrophils, low-density granulocytes,which have an enhanced capacity to form NETs(38,39). NETs can amplify inflammatory responses in

CENTRAL ILLUSTRATION Progression of Vascular Inflammation in Human Inflammatory Models

Teague, H.L. et al. J Am Coll Cardiol. 2017;70(11):1403–12.

A gross illustration of the aortic arch has been taken in cross section to magnify the vessel wall. Neutrophil activation due to systemic inflammation leads to

the formation of neutrophil extracellular traps and may initiate damage to the endothelium. Monocytes and T cells then infiltrate the lesion. Monocytes

differentiate into macrophages, where they proliferate to sustain their population. Macrophages within the vessel wall have high glycolytic rates and take up the18F-fluorodeoxyglucose (FDG) tracer, which is detectable by FDG positron emission tomography in human models of inflammation. IFN ¼ interferon; IL ¼ interleukin;

TNF ¼ tumor necrosis factor.

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the plaque and other tissues, including inflamma-some activation in macrophages and induction oftype I IFN synthesis by plasmacytoid dendritic cells(38,41).

Two pathways are of potential interest in targetingvascular risk in SLE: the inhibition of type 1 IFNs andthe blockage of NETosis through PAD-4 inhibition.Type 1 IFNs signal through the JAK/STAT pathway,which is inhibited by tofacitinib. Tofacitinib amelio-rated vascular dysfunction in a murine lupus model(42) and is being tested in humans in an ongoingclinical trial (NCT02535689). Inhibition of pathwaysimplicated in NET formation using chemical in-hibitors of PAD-4 leads to amelioration of endothelialdysfunction, a prothrombotic phenotype, and plaque

formation in murine models of lupus and athero-sclerosis (41). Overall, SLE represents a unique modelfor understanding the role of the innate immunesystem in the development of vascular disease, andmay allow the characterization of novel therapeutictargets in subsets of patients.

PET IMAGING OF THE ARTERY WALL

Currently, FDG PET imaging is a cost-effective clinicalstandard of care for diagnosis, staging, and moni-toring the response of many malignancies, and itsrole is significantly enhanced by the introduction ofPET/CT imaging and PET/MR imaging. Imaginghuman inflammation models with positron emission

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tomography allows investigations to be conductedwithout expensive animal models, a step towardclinical translation.

PET imaging of VI using FDG was first describedmore than 15 years ago (43,44) and has becomeimportant in atherosclerosis research. FDG accumu-lates within living cells in proportion to their glyco-lytic rates (45). Several tissues have particularly highglycolytic rates and hence tend to accumulate FDG,including tumors, brain tissue, myocytes, andinflammatory cells. Phagocytes have particularly highglycolytic rates, especially after proinflammatoryactivation (46), leading to high FDG retention. Apotential advantage of PET imaging in addition toother available anatomic imaging techniques (e.g., CTimaging, MR, and ultrasound) is through the ability toadminister miniscule concentrations of a substancethat can be targeted for a specific molecular process.PET images can be interpreted both qualitatively andquantitatively to evaluate early phases of disease orshort-term changes in VI related to treatment thatmay not have concordant changes in morphology.Several studies show that FDG uptake, whenmeasured in the arterial wall in vivo, reflects the levelof macrophage accumulation within the atheroma(47). Atherosclerotic lesions show higher VI by FDGand tend to experience greater subsequent progres-sion (48). Moreover, the FDG measurement of VI mayprovide an independent index of the risk for subse-quent CV events (48–50), which is the subject oflarger ongoing prospective studies (51) to evaluate thestandardization of ideal imaging procedures (52,53)and subsequent clinical application.

FDG imaging of VI is increasingly used to evaluatetherapeutic compounds targeting atherosclerosis.The best therapies studied in this regard are statins,which show a reduction in the arterial FDG signalconsistent with their impact on CV events in ran-domized clinical trials (54,55). However, a lack ofreduction in arterial FDG uptake associates with aparallel failure to reduce CV events for a number ofexperimental treatments (56–58). Prospectiveoutcome data and response to novel therapies usingFDG in vascular diseases are limited. However, giventhe general concordance between VI imaging andCVD outcomes, relatively small and swift FDG imag-ing studies may facilitate drug discovery.

Although FDG imaging adequately addressescertain pathophysiological and treatment-relatedquestions, the specificity for inflammation with thisagent is not clearly defined, because of the variableaffinity for glucose of all cells in the body. Addition-ally, high FDG activity in the blood pool and tissuesnear the vessel wall complicates quantification. Thus,

there are opportunities for other imaging agents foratherosclerosis, such as vascular calcification imagedwith 18F–sodium fluoride (NaF). Originally approvedby the U.S. Food and Drug Administration for boneimaging in oncology, NaF PET imaging is an emergingtechnique reputed to capture a different aspect of thedevelopment of atheroma compared with FDG (59),related to calcification and microcalcification withinan atherosclerotic plaque (Figure 3). In patients withMI and stable angina, Joshi et al. (59) demonstratedconspicuous uptake of NaF in plaque, with histolog-ical evidence of macrophage infiltration, calcification,necrosis, and apoptosis, as well as high-risk plaquefeatures imaged by intravascular ultrasound. Advan-tages of NaF over FDG include rapid backgroundblood and tissue clearance and absence of activity inthe myocardium, improving the potential for imagingpathological NaF uptake in the coronary arteries (53).As with FDG, elements of the cell/process specificityof NaF in atherosclerosis have been described, andimaging technique standardization is an active realmof research.

In parallel with further FDG and NaF work, inves-tigation continues with other PET, CT, and MR tech-niques to study varied mechanisms and applicationsfor CVD imaging. An abbreviated list of PET agentscurrently under investigation is shown in Table 1.Agents from the list that already have Food and DrugAdministration approval for human imaging ofnonvascular processes include 68Ga-DOTA-octreotateand 18F-florbetapen. 68Ga-DOTA-octreotate has highaffinity for somatostatin receptors that are present inhigh concentration on inflammatory leukocytes (60).Its low background activity and high signal make it apromising candidate for plaque imaging, and itsfeasibility in humans has been demonstrated incomparison with FDG (60). 18F-florbetapen is a PETagent that is used clinically to characterize b-amyloidplaque in neurodegenerative processes, such as Alz-heimer’s disease. Bucerius et al. (61) demonstratedthat higher carotid artery uptake of 18F-florbetapen isassociated with male sex, independent of centralnervous system uptake, which potentially implicatesthe presence of higher concentrations of inflamma-tory b-amyloid within the vessel wall. Further in-vestigations of the performance characteristics ofthese new agents are ongoing.

In summary, with a new understanding of immu-nologic processes applied to the established feasibilityof promising PET agents for imaging atherosclerosisin humans, we now can administer these techniquesin newly identified human disease models to betterunderstand atherogenesis (Central Illustration).Acknowledging the great potential for pathology-

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specific and quantitative information that PET imag-ing has for atherosclerosis, an outcome-based multi-disciplinary approach for further investigation,integrating biology, engineering, imaging physics, andeconomics, will continue to help us understand howthese techniques ultimately fit into clinical care andtranslational research.

CONCLUSIONS

Inflammation has emerged as a critical factor in earlyatherosclerosis. An understanding of the immunecells involved in the initiation and progression ofVI may facilitate the identification of important

pathways to target for future CVD therapeutics.Furthermore, the use of PET imaging to measure VImay improve our ability to identify the most prom-ising therapies to take to phase III clinical trials. Assuch, advances in immunology and vascular imaginghave the potential to accelerate discovery of newtreatments to reduce the burden of CVD.

ADDRESS FOR CORRESPONDENCE: Dr. Nehal N.Mehta, Cardiovascular and Pulmonary Branch,National Heart, Lung, and Blood Institute, 10 CenterDrive, CRC, Room 5-5140, Bethesda, Maryland, 20892.E-mail: nehal.mehta@nih.gov.

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KEY WORDS cardiovascular imaging,monocytes, neutrophils, T cells