Atherosclerosis

review
F o c u s o n Va s c u l a r D i s e a s e
Atherosclerosis: current pathogenesis and therapeutic options

Christian Weber13 & Heidi Noels4
Coronary artery disease (CAD) arising from atherosclerosis is a leading cause of death and morbidity worldwide. The underlying pathogenesis involves an imbalanced lipid metabolism and a maladaptive immune response entailing a chronic inflammation of the arterial wall. The disturbed equilibrium of lipid accumulation, immune responses and their clearance is shaped by leukocyte trafficking and homeostasis governed by chemokines and their receptors. New pro- and anti-inflammatory pathways linking lipid and inflammation biology have been discovered, and genetic profiling studies have unveiled variations involved in human CAD. The growing understanding of the inflammatory processes and mediators has uncovered an intriguing diversity of targetable mechanisms that can be exploited to complement lipid-lowering therapies. Here we aim to systematically survey recently identified molecular mechanisms, translational developments and clinical strategies for targeting lipid-related inflammation in atherosclerosis and CAD.
Atherosclerosis gives rise to cerebrovascular disease and CAD through a slowly progressing lesion formation and luminal narrowing of arteries. Upon plaque rupture and thrombosis, these most common forms of cardiovascular disease manifest as acute coronary syndrome (ACS), myocardial infarction or stroke. The underlying pathology is characterized by a chronic inflammatory process of the arterial wall that occurs at predilection sites with disturbed laminar flow, such as branch points1. It is initiated by endothelial dysfunction and structural alterations, including the absence of a confluent luminal elastin layer and the exposure of proteoglycans2, which permit subendothelial accumulation of low-density lipoprotein (LDL). Elevated levels of circulating cholesterol transported by apolipoprotein B100 (ApoB100)-containing LDL promote atherosclerosis and cardiovascular disease3. ApoB100 binding to negatively charged extracellular matrix proteoglycans leads to retention of LDL particles in the intima, where they are susceptible to oxidative modification by reactive oxygen species or enzymes such as myeloperoxidase or lipoxygenases released from inflammatory cells. Oxidized lipids and LDL (oxLDL) trigger the expression of adhesion molecules and the secretion of chemokines by endothelial cells, which, together with the deposition of platelet-derived chemokines, drive intimal immune cell infiltration. Early fatty-streak lesions consist of T cells and monocyte-derived
for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany. 2Munich Heart Alliance, Munich, Germany. 3Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands. 4Institute for Molecular Cardiovascular Research, Rheinisch-Westflische Technische Hochschule Aachen University, Aachen, Germany. Correspondence should be addressed to C.W. (christian.weber@med.uni-muenchen.de). Published online 7 November 2011; doi:10.1038/nm.2538
1Institute
2011 Nature America, Inc. All rights reserved.
macrophage-like foam cells loaded with lipids. Successive accumulation of apoptotic cells, debris and cholesterol crystals forms a necrotic core. Fibroatheromatous plaques are covered by a fibrous cap composed of collagen and smooth muscle cells (SMCs), which are replaced by macrophages in the thinning inflamed caps that are prone to rupture. The shoulder regions are heavily infiltrated by T cells and mast cells, which produce enzymes and proinflammatory mediators, contributing to adventitial inflammation of advanced plaques4. Hypercholesterolemic apolipoprotein Edeficient (Apoe/) or LDL receptordeficient (Ldlr/) mice are frequently used to study mechanisms of disease initiation and progression but are less suitable for investigations of advanced stages, such as plaque rupture or thrombosis, which continue to rely on histopathological and clinical studies35. Given the undisputed limitations of studying atherosclerosis in mice, one needs to apply caution when extrapolating mechanisms or therapeutic options to human disease, unless reproducible cross-species studies are implemented for validation. The atherosclerosis field is so vast that attempts to comprehensively cover all aspects will be futile. Whereas the roles of monocytes6, macrophages1 and innate versus adaptive immunity3 have been extensively discussed, this review focuses on new aspects of monocyte and macrophage recruitment and on hitherto less appreciated cellular players, namely neutrophils and dendritic cell (DC) subtypes. It further aims to scrutinize the intriguing diversity of lipid-related proinflammatory and atheroprotective pathways affecting the disease equilibrium, to review genetic variants contributing to atherosclerosis and to discuss clinical applications of targeting inflammation in atherosclerosis and CAD. Chemokines in atherogenic cell recruitment and homeostasis Atherogenic recruitment of leukocytes involves a sequence of rolling, firm adhesion, lateral migration and transendothelial diapedesis
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Figure 1 Roleofchemokinesandtheirreceptors Chemokine (receptor) functions inatherogenesis.Leukocyterecruitmentinvolves Lumen theirrolling,firmadhesion,lateralmigration Cell homeostasis Cell recruitment andtransendothelialdiapedesis,controlled bychemokines.Whereassolublechemokines CX3CR1 Tethering Firm adhesion Lateral GPCR transmigration CX3CL1 mediatedirectleukocyterecruitment, and rolling and diapedesis Ly6Clow monocyte chemokinesimmobilizedonthesurfaceof GAG Integrin Cell survival Chemokine activatedendothelialcells(ECs)through egress? SELPLG CCL17 glycosaminoglycan(GAG)triggerleukocyte arrestthroughGproteincoupledreceptors Treg cell homeostasis ? ICAM-1 (GPCRs),whichactivateleukocyteintegrins. SELP VCAM-1 Endothelium Cell-dependentandlesion-stagedependent SELE usageofchemokinesandtheirreceptors12show Proinflammatory therobustnessandcomplexityofthechemokine cytokine system,providingasignaturecombinationof chemokinesineachstagetoattractspecific Aortic transplant model Cell-dependent actions Synergistic actions leukocytesubsets.Thecombinedactionof low Ly6Chigh Neutrophil T cell Ly6C Monocyte chemotaxis chemokinesinleukocyterecruitmentcanresult insynergisticeffects,asfortheCCL5-CXCL4 heteromer23.Whetherchemokinescanalso CCR7 CCR2 Lesion CCR5 CX3CR1 CXCR2 CXCR3/6 DC-like mediatecellegressfromatheroscleroticlesions CCR1/2/5 CCR5 CCR5 CCL5 CXCL4 remainsunclear27andcouldbeinherenttothe Healthy aortictransplantationmodelstudied26,28.Some Lymph node chemokinesandtheirreceptorsareinvolvedin Lesion-stagedependent actions cellhomeostasis,suchasCX3CL1andCX3CR1 Potential therapies Initial Early Advanced (ref.24)andCCL17(ref.25)inmonocyteand CCL5 CCL2 Modulating GAG Small-molecule Manipulating CCR2 Tregcellhomeostasis,respectively.Interference binding and receptor receptor antagonists heteromeric chemokine CXCR3 CCR5 CX3CR1/CX3CL1 withchemokinefunctionsincreasinglygains or blocking Ab interactions affinity of chemokines attentionincardiovasculardrugresearch (blueboxes)7.Blockingchemokines(ortheir receptors)withsmall-moleculeantagonistsorantibodiesormodulationofchemokineaffinityforreceptorsorofGAGbindingcanreducechemokine signaling.Tailoredmanipulationofheterophilicchemokineinteractionsmayevenbemorepromising,asitcanleavenormalphysiologicaland immunologicalfunctionsintact.SELPLG,selectinPligand;SELP,selectinP;SELE,selectinE;ICAM-1,intercellularadhesionmolecule1;VCAM-1, vascularcelladhesionmolecule1.
and is controlled by chemokines, which are chemotactic cytokines classified according to their conserved cysteines, and their correspondingly categorized G proteincoupled receptors. The various leukocytes recruited during the inflammatory cascade encompass neutrophils, monocytes and T cells, but also B cells, DCs and mast cells5. Considering the diversity of these cell subsets and their functions, the abundance in the chemokine system has been perceived to confer robustness and specificity7. At a site of inflammation, particular leukocyte subsets may be recruited by a signature combination of chemokines engaging in heterophilic interactions, facilitated by a local repertoire of proteoglycans with differential binding affinities for chemokines7. Studies using gene-deficient mice and antagonists have generated valuable insights into chemokine ligand-receptor axes and their specific and combined contributions to atherogenesis7. Specific functions and combined interference. The possibility of disrupting complementary chemokine functions has been explored along the paradigm of sequential monocyte mobilization and influx (Fig. 1). In mice, classical Ly6Chigh cells dominate hyperlipidemia-induced monocytosis and employ the chemokine receptors CCR2, CCR5 and CX3CR1, to invade plaques, where they differentiate into macrophages, whereas entry of patrolling Ly6Clow monocytes occurs less frequently and seems to rely on CCR5 (refs. 8,9). Genetic deletion of CCL2 and CX3CR1, or CCR2 and CX3CL1 decreases atherosclerosis compared with single deficiencies in these proteins, which is attributable to both attenuated hyperlipidemia-associated blood monocytosis and reduced macrophage accumulation 10,11. The CCL5 receptor antagonist Met-RANTES has been shown to further lower circulating monocyte counts, almost fully protecting against atherosclerosis10. Thus, combined blockade of chemokine receptors seems more effective
than single blockade or deficiency, but the different rate-limiting contributions to individual steps of mobilization, recruitment and homeostasis, and the specific atherogenic functions of monocyte subsets, remain to be clarified. Temporal patterns of chemokine expression and function at distinct stages of atherogenesis have been addressed12 (Fig. 1). Genetic deletion of CCR2 in mice did not prevent early lesion formation in the abdominal aorta but rather limited late-stage plaque formation and monocyte infiltration in the aortic root12,13. In contrast, mouse CXCR3 deficiency primarily delayed early atherosclerosis by attenuating recruitment of T helper 1 (TH1) T cells, while increasing the number of regulatory T cells (Treg cells)13 within the lesion. The atheroprotective role of FoxP3+ Treg cells producing anti-inflammatory interleukin-10 (IL-10) and transforming growth factor- has been established by transfer experiments3, and shifting T cell responses in favor of Treg cells has been achieved by disrupting the CXCR3-CXCL10 axis14,15. The lack of the CCL5 receptor CCR5 in somatic or blood cells reduced plaque formation in mice at later stages, upregulated systemic IL-10 and downregulated interferon- (IFN-), which reflects an attenuated TH1-type response. This is in contrast to deficiency of CCR1, the other major CCL5 receptor, and indicates that CCR5 deficiency is protective by modulating the immune balance rather than limiting cell influx16,17. The combined action of chemokines in leukocyte recruitment is illustrated by CCL5 and CXCL4 (Fig. 1), which are deposited by platelets or platelet-derived microparticles during their transient P-selectinmediated interactions on inflamed endothelium, where they promote atherogenic monocyte arrest18,19. This event was linked to lesion formation, which is exacerbated by repetitive injections of activated platelets18 but inhibited by genetic deletion of CXCL4 or
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treatment with Met-RANTES20,21. Interestingly, CXCL4 amplifies CCL5-triggered monocyte arrest22, and inhibition of this synergism by blocking CCL5-CXCL4 heteromerization reduces atherogenesis in hyperlipidemic mice23. Role in homeostasis and regression. Aside from the functions of chemokines in recruitment, the absence of CX3CR1 or CX3CL1 has been show to reduce Ly6Clow blood monocyte levels under steadystate and inflammatory conditions, which could be rescued by expression of antiapoptotic Bcl2, indicating that this axis confers essential survival signals24. Enforced survival of monocytes, plaque phagocytes and foam cells restored atherogenesis in Cx3cr1/ mice, emphasizing homeostatic functions of this axis in atherosclerosis. Conversely, deficiency or antibody blockade of DC-derived CCL17 in hyperlipidemic mice reduced atheroprogression by expanding Treg cells, whereas CCL17 expression in DCs restricted Treg cell maintenance to promote atherosclerosis25, identifying CCL17 as a central regulator of Treg cell homeostasis (Fig. 1). A role of chemokines in plaque regression is insufficiently characterized because of the lack of proper models. Plaque regression has been accomplished by transplantation of diseased aortas into normolipidemic mice, revealing an upregulation of CCR7. Blocking antibodies to CCR7 ligands inhibited regression, implicating CCR7driven cell egress as an underlying mechanism26. However, in a model of virus-mediated Apoe gene transfer, no involvement of CCR7 was evident, and reduced mononuclear cell influx combined with apoptosis, rather than cell emigration, was held responsible for plaque regression27. Altered inflammatory properties of plaque monocyte-derived cells with high CCR7 expression and CCR7-driven cell emigration promoted by liver X receptor (LXR) agonism or rapid high-density lipoprotein (HDL) normalization to cause regressionmay thus be related to the aortic transplantation model28 (Fig. 1). Indeed, somatic CCR7 deficiency reduced atherosclerosis in Ldlr/ mice by impairing CCR7-dependent T cell priming and recirculation between inflamed lesions and secondary lymphoid organs29. Notably, neutralizing the noncanonical chemokine macrophage migration inhibitory factor (MIF) caused regression of established lesions, probably because of its CXCR2 and CXCR4 agonist effect, which promoted macrophage and T-cell infiltration30. The identification of mediators of cell influx into advanced lesions will be crucial to open avenues for a causative treatment of atherosclerosis. Neutrophils: initiators of innate immunity in atherogenesis In the arterial wall, defense against pathogenic damage requires a rigorous sentinel system of endothelium, patrolling monocytes, resident macrophages and DCs and involves pattern recognition receptors (PRRs) sensing damage-associated molecular patterns (DAMPs) to mount an innate immune response31. Recent evidence has emerged to suggest an important role for neutrophils in the initial phase of atherosclerosis in mice. Early neutrophil inflammatory signals trigger intimal recruitment of monocytes, which differentiate into macrophages and internalize native and modified LDL, leading to foam cell formation. Macrophages can amplify LDL modification, promote vulnerability of advanced plaques through secretion of cytokines, proteases or procoagulant factors, undergo apoptosis and, when not effectively cleared, contribute to necrotic core formation. Innate immune signaling by modified LDL and atherogens. As they differentiate, macrophages upregulate scavenger receptors (such as SR-A/B and CD36), which are multifunctional PRRs, clearing cell debris, recognizing oxidation-specific epitopes and also internalizing modified LDL. In addition, endothelial cells, lesional macrophages and DCs express diverse Toll-like receptors (TLRs), another type of PRR mediating inflammatory activation. Like pathogen-derived endotoxins, modified LDL can trigger TLR2 and TLR4 signaling through CD36 (refs. 32,33), whereas genetic deletion of TLR2, TLR4 or the TLR signaling adaptor MyD88 reduces atherosclerosis32. By analogy to pathogen pattern recognition, patrolling monocytes or tissueresident cells monitor and recognize alarm signals, including modified lipids or components expelled by damaged cells. High-mobility group box-1 protein (HMGB1), a chromatin protein from necrotic cells, induces macrophage synthesis of cytokines and chemokines promoting neutrophil recruitment through interactions of complexes formed by HMGB1 and DAMPs, such as endotoxin or singlestranded DNA, with the canonical HMGB1 receptor RAGE or TLRs 2, 4 and 9 in a Myd88- and nuclear factor-B (NF-B)-dependent pathway31 (Fig. 2). Notably, HMGB1 neutralization reduces dietinduced atherosclerosis in mice34. Hence, atherogens and pathogens may use a similar immune receptor repertoire. Neutrophils: emerging players in atherosclerosis. Polymorphonuclear leukocytes (neutrophils) have been detected in aortic fatty-streak lesions of primates and have been correlated with CAD incidence and severity5; however, convincing evidence for a proatherogenic role of neutrophils has been elusive until recently. Neutrophils and their mediators have been detected in human and mouse atherosclerotic lesions5,35,36. Although mice have a myeloid-biased immune system compared to humans, neutrophils represent about 2% of lesional leukocytes and accumulate in regions with high monocyte density in mice36. Neutrophils use the CCL5 receptors CCR1 and CCR5 for entering atherosclerotic plaques, compared to a restricted use of CCR2 and CXCR2 for peripheral venous recruitment37. A disturbed lipid balance facilitates their recruitment to lesions, as shown in bone marrow ABC transporter-deficient mice, which showed defective cholesterol efflux, increased TLR signaling and inflammatory gene expression, and marked neutrophil infiltration upon peripheral inflammatory signals38. Notably, hyperlipidemia triggers neutrophilia by stimulating granulopoiesis and bone marrow egress, involving elevated CXCL1 plasma levels, and the amount of circulating neutrophils closely correlates with plaque size37. Disrupting the CXCR4-CXCL12 axis increases peripheral neutrophil counts with immature forms and aggravates lesion formation, whereas neutrophil depletion predominantly reduces early plaque size35,37, indicating a functional role of neutrophils in murine atherosclerosis. Role in onset, progression and resolution of inflammation. During extravasation, neutrophils sequentially release preformed granule proteins (Fig. 2). For instance, cationic azurocidin is deposited on the endothelium, triggering monocyte arrest and upregulating endothelial adhesion molecules31, and cathepsin G and LL-37 (also known as CAMP) attract monocytes by activating formyl-peptide receptors. Notably, neutrophil depletion specifically decreases classical monocyte recruitment in mice, an effect rescued by neutrophil-secreted LL-37 and azurocidin39. In the vascular wall, oxLDL may induce neutrophil transmigration and degranulation, which may then trigger rapid recruitment of classical monocytes usually present in hyperlipidemia-induced atherosclerosis. As short-lived cells, neutrophils rapidly become apoptotic, also in the intima35, releasing a cocktail of find-me and eat-me signals to attract monocytes/ macrophages for scavenging31. Among resolution signals released
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Figure 2 Neutrophilsascrucialplayersin atherogenesis.Hyperlipidemiaincreasesthe numberofcirculatingneutrophils,whichare recruitedintoatheroscleroticlesionsthrough specificchemokinereceptors37.Macrophage activationthroughatherogeniclipids33,a disturbedintracellularlipidbalance82andDNA releasedfromapoptoticcellscantriggerreleaseof cytokinesandneutrophil-attractingchemokines. OxLDLmayinduceneutrophiltransmigration andreleaseofinflammatoryreactiveoxygen species(ROS)andgranuleproteins,whichtrigger monocyterecruitmentandextravasationdirectly orindirectlythroughupregulationofadhesion moleculesonendothelialcells.Apoptotic neutrophilssustainmonocyterecruitment throughvariousfind-meandeat-mesignals31. Furthermore,NETformationuponneutrophil activation42andneutrophilprotease-mediated proteolysisofthetissuefactorpathwayinhibitor44 couldpromoteatheroprogressionandthrombus growth,respectively.Altogether,neutrophils couldprovideachronicinflammatorytrigger sustainingatherogenesis,despitetheirability toprovideresolutionsignals31thatmaytrigger antiatherogenicTLR3signaling40.ssDNA,single stranded-DNA;RAGE,receptorforadvanced glycosylationendproducts.
Neutrophil-mediated monocyte recruitment Neutrophil Neutrophil Recruitment LDL CCR1/2/5 CXCR2 Find me TLR4/6 Chemokines Cytokines ROS
Granule proteins (azurocidin, cathepsin G, LL-37)
NET Thrombus formation Neutrophil activation
Monocyte high (Ly6C )
CD36
Apoptotic neutrophil
TFPI
HMGB1 DNA ssDNA RNA
Macrophage Lipid raft associated receptor signaling TLR9 RAGE TLR Excess free cholesterol oxLDL ssDNA
Cathepsin G Nucleosomes neutrophil elastase
RNA
Eat me Resolution signals Anti-inflammatory Scavenging
TLR3
by damaged neutrophils to resolve inflammation, RNA may enhance TLR3 signaling to delay inflammatory lesion formation, as evident by increased atherosclerosis in TLR3-deficient Apoe/ mice40 (Fig. 2). Neutrophils as chronic atherogenic triggers? It will be crucial to identify determinants that lead to chronic inflammation in atherosclerosis. Notably, intimal macrophages undergo apoptosis, become necrotic during progressive atherosclerosis and finally coalesce into the necrotic core of vulnerable plaques, probably owing to defective phagocytic clearance (efferocytosis) of apoptotic cells overstraining the efferocytotic mediators MFG-E8, C1q and Mertk41. Furthermore, neutrophils activated through TLRs, Fc receptors or cytokine receptors can release nuclear content (DNA fibers, chromatin) that forms a scaffold containing antimicrobial proteins, such as LL-37, termed neutrophil extracellular traps (NETs)42 (Fig. 2). Given the prominent role of CXCR2 as a proatherogenic receptor on both monocytes and neutrophils12, the fact that LL-37 can act as a CXCR2 ligand43 indicates that NET formation involving LL-37 may participate in chronic atherogenesis. A link between neutrophils and coagulation has been provided by evidence that neutrophil serine proteases along with externalized nucleosomes promote intravascular thrombus growth in vivo by enhancing tissue factor and factor XIIdependent coagulation through proteolysis of the tissue factor pathway inhibitor44 (Fig. 2). Without a pathogen challenge, the continued presence of neutrophils in advanced plaques may thus contribute to large-vessel thrombosis as a trigger for myocardial infarction and stroke. Dendritic cells at the cross-roads to adaptive immunity The importance of antigen-specific immune responses in atherogenesis is widely acknowledged3. By sensing atherogenic danger signals and antigen internalization, DCs may be ideally positioned at the crossroads of innate and adaptive immunity and may be instrumental for orchestrating a switch from tolerance to adaptive immunity activation5.
T cells and B cells as effectors in atherogenesis. Atherosclerotic plaques feature a clonal expansion of memory-effector T cells, which indicate an antigen-specific reaction, and can also confer lesion formation upon reconstitutive transfer3. Moreover, activation of CD8+ T cells against an SMC-expressed artificial antigen exacerbates atherosclerosis in hyperlipidemic mice45. Ldlr/ mice deficient in the major TH1-differentiating transcription factor T-bet show a reduced lesion size46, indicating that atheroprogression is driven by a TH1-type response and its signature cytokines IFN-, IL-12 and IL-18 (ref. 3). Conversely, numerous studies have established atheroprotective effects mediated by Treg cell subsets and their products transforming growth factor- and IL-10 (ref. 3), which can be induced by administration of exogenous immune modulators or oral CD3-specific antibody47,48. In contrast, data for TH2 and TH17 cells are less conclusive and partially contradictory3. Immunization studies have mostly focused on modified LDL and neoepitopes generated by oxidation; however, vaccination with native LDL and its peptides has also proved successful49. The standing hypothesis that LDL-reactive T cells are eliminated by thymic negative selection has been challenged by findings that immunization against an ApoB100-recognizing T cell receptor diminished antigen-specific T cell responses and atherosclerosis50. This indicates the pathogenic character of native LDL-reactive T cells and may be explained by minimal LDL modifications or incomplete tolerance to autoantigens, which under normal conditions may be controlled by peripheral mechanisms. Regarding B cells, bone marrow B cell deficiency or splenectomy aggravates atherosclerosis in mice, whereas splenic B cell transfer mediates protection in splenectomized recipients3. Accordingly, deficiency in bone marrow IL-5, a cytokine that expands natural B-1 cells, increases atherosclerosis and reduces oxLDL-reactive IgM levels 51. Although oxLDL-specific IgG titers correlate with CAD52, oxLDL-specific IgM titers are associated with atheroprotection49, whichlike regressioncan be conferred by recombinant antibodies to oxLDL in mice53. Notably, depletion of conventional
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Figure 3 DCsatthecrossroadsofadaptive Monocyte Limited Treg immunity.DCsmaybeideallypositionedatthe high Treg cell low Ly6C Ly6C maintenance crossroadsofinnateandadaptiveimmunity, + LDL CD4 T cell + translatinginnatedamagesignals,suchas low CD11c Ly6C high Ly6C EC oxLDL,intoanenhancedadaptiveimmune response.ResidentintimalCD11c+DCs Chemokine CCL17 arepresentinatherosclerotic-proneregions ~25% ofhealthyvessels5658throughCX3CR1~60% relatedmechanisms60,andCD11c+DC + T cell CD4 T cell ? + + numbersincreaseuponlesionformationand CD11c DC recruitment CCL17 DC activation Macrophage DC progression25,59.MainlyLy6Clowbutalso proliferation Apoptotic/ Ly6Chighmonocytes9giverisetoCD11c+cellsin necrotic Pro cell pDC oxLDL inflammatory lesions,andGM-CSFmayinduceDCproliferation CX3CR1 cytokines DNA intheintima62,63.Althoughdiversecombinations Foam cell ssRNA + CD11c ofsurfacemarkershavebeenusedtoidentify GM-CSF Foam cell formation resident TLR7/9 lesionalDCs,atruedistinctionbetween intimal DC Proinflammatory macrophagesandDCsremainsdifficultbecause IFN- ? cytokine oftheirplasticity.LesionalDCstakeuplipids, DC homeostasis production IFN- TRAIL produceproinflammatorycytokinesandpolarize + Proinflammatory CD4 T cell IFN- CD4+TcellstoatherogenicTH1responses,all cytokine activation;TH1 Cytotoxic T cell effector functions + SMC production polarization CD11c DC contributingtotheirproatherogeniccharacter. Inadvancedplaques,CCL17+DCslimitTregcell Advanced lesion expansionandexertlocalimmune-regulatory Healthy Atherosclerotic vessel functionsbyrecruitingandactivatingCD4+ Tcells25.Moreover,pDCsandpDC-derivedIFN- canbefoundinadvancedplaques,wheretheycontrolcytotoxicTcelleffectorfunctionsbystimulatingnaiveCD4+TcellstoexpressIFN-andTRAIL69 andtriggerantigen-presentingcellstoproduceproinflammatorymediators68.TRAIL,tumornecrosisfactorrelatedapoptosis-inducingligand.
B-2 cells ameliorates atherosclerosis, whereas transfer of B-2 but not B-1 cells promotes atherosclerosis, confirming subsetspecific effects54,55. From lipid homeostasis to antigen sensing: DC versatility. DCs are a heterogeneous population that includes conventional DCs (cDCs) subdivided into lymphoid-tissue-resident or migratory subtypes, plasmacytoid DCs (pDCs) and inflammatory DCs, which are not found in steady states5. Although DCs are bona fide antigen-presenting cells, they can also engulf lipids, sharing phenotypic and functional features with macrophages. The exact distinction between macrophages and DCs thus remains controversial and confounded by their plasticity. A DC-based network is detectable in the intima of healthy human arteries56 and in the aorta of CD11c-EYFP reporter mice, where they represent 0.5% of aortic cells and mainly localize to atherosclerosis-prone regions57,58. Mature DCs further accumulate during atheroprogression and cluster with T cells in the shoulder and rupture-prone plaque regions5,25,59 (Fig. 3). During low-grade chronic inflammation, vascular DCs accumulate by recruitment of blood-borneprobably Ly6Clowmonocyte precursors through a process involving CX3CR1 (refs. 24,57,60). Accordingly, Ly6Clow monocytes express CD11c in severe hyperlipidemia, promoting atherosclerosis and intimal accumulation of CD11c+ monocytes and macrophages61. The preferentially recruited Ly6Chigh monocytes can also give rise to CD11c+ DCs, and granulocyte-macrophage colonystimulating factor (GM-CSF) can drive lesional DC content and plaque growth, probably through stimulating intimal DC proliferation62,63 (Fig. 3). Resident intimal CD11c+ DCs accumulate lipids64, which can occur through SRmediated internalization or capturing of circulating lipoproteins with luminal dendrites. Notably, dyslipidemia activates DCs to inhibit their migration to lymph nodes for peripheral sequestration65. Prolonging the DC lifespan by human Bcl2 expression resulted in increased cDC numbers and reduced plasma cholesterol levels, whereas short-term depletion of DCs in Apoe/ mice increased hypercholesterolemia66. This implicates cDCs in cholesterol homeostasis and proatherogenic
effects, as illustrated by their depletion64 and reduced intimal aortic CX3CR1+ DCs numbers and lesion size in Cx3cr1/ mice60. But DCs also control early atherogenic immune activation (Fig. 3). In bioengineered arteries, endotoxin-activated adventitial myeloid DCs stimulate autologous CD4+ T cells to produce IFN- and infiltrate the arterial wall, breaking T cell tolerance to self antigens and initiating inflammation67. Similarly, aortic CD11c+ DCs can trigger antigen-specific T cell proliferation, corroborating that intimal DCs can internalize antigen to specifically prime T cell responses 25,58. In advanced plaques, CCL17 expression by mature DCs restricts T reg cell expansion and sustains atherosclerosis, identifying the control of Treg cell homeostasis as an effector mechanism of CCL17+ DCs in atherogenesis25. Moreover, CCL17+ DCs can interact with CD4+ T cells in atherosclerotic arteries, indicative of local immuneregulatory functions25. pDCs, which are a phenotypically distinct DC subtype secreting type I IFNs after TLR7 or TLR9 stimulation5, cluster with myeloid DCs in atherosclerotic shoulder regions68 (Fig. 3). pDC-derived IFN- correlates with plaque instability and can regulate cytotoxic T cell effector functions by stimulating naive CD4+ T cells to express IFN- and TRAIL, enabling them to kill vascular SMCs69. Moreover, pDCs act as inflammatory amplifiers, as antigen-presenting cells respond to IFN- by producing proinflammatory mediators68. Sensing of microbial pathogens by pDCs may link atherosclerosis to infectious burden, explaining plaque vulnerability during acute infections69. Likewise, nucleotides or DNA released from necrotic or apoptotic cells in plaques may activate pDCs through TLR7 and TLR9. Hence, pDC may translate innate damage signals into an enhanced adaptive immune response. Atherogenic signature inflammatory mediators Unleashed inflammatory signaling pathways may be attractive targets for controlling atherosclerosis. As important myeloid effector cytokines, type I IFNs have been implicated in atherogenesis. Treatment of atherosclerosis-prone mice with IFN- increases lesion size, macrophage content and CCL5 plasma levels, whereas myeloid deficiency of IFNAR1, the receptor for type I IFNs, attenuates
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atherogenesis, macrophage accumulation and necrotic core formation70. Signaling through IFN-IFNAR1signal transducer and activator of transcription (STAT) triggers CCL5 production by macrophages, increasing CCR5-mediated monocyte recruitment70. This explains a correlation between upregulated type I IFN signaling and CCL5 expression in advanced human lesions70. Human atherosclerotic lesions, especially of advanced stage, show an upregulation of MIF in macrophages and endothelial cells, which express MIF in response to oxLDL. Multiple mouse studies have confirmed the atheroprogressive function of MIF, which is probably linked to its versatile proinflammatory properties at crucial checkpoints in atherogenesis, such as endothelial cell activation, monocyte recruitment, macrophage and foam cell formation, SMC migration and extracellular matrix destabilization71. True regression of atherosclerotic lesions in mice receiving MIF-specific antibody suggests MIF as a promising drug target30,71. Co-stimulatory and co-inhibitory signaling molecules fine-tune immune reactions by regulating inflammatory phenotypes and responses of T cells and antigen-presenting cells. The B7-CD28 and TNFTNF receptor families have been implicated in different stages of atherogenesis, either in a stimulatory or an inhibitory way72. Unraveling the complex cell type and stage-specific interplay of coregulatory pathways may offer interesting therapeutic options72. For the prominent co-stimulatory dyad CD40-CD40L, both receptor and ligand are expressed by lesional cell types and highly upregulated in advanced human plaques73. Mouse studies have revealed a plaquedestabilizing function for CD40-CD40L signaling, attributable to the induction of multiple inflammatory factors73. Notably, repeated injection of CD40L-deficient platelets specifically implicated platelet CD40L in atherogenesis by mediating proinflammatory leukocyteplatelet interactions and Treg cell homeostasis without compromising systemic immune responses or inducing hemorrhage74. Thus, cellor pathway-specific interventions in CD40-CD40L signaling75 hold promise as cardiovascular therapeutics. Linking lipids and inflammation in atherogenesis Foam cell formation involves phagocytosis of matrix-retained lipoproteins and fluid-phase pinocytosis of aggregated lipoproteins by macrophages1, but substantial evidence also suggests that oxidative modification of subendothelially accumulated LDL triggers the inflammatory process underlying atherosclerosis. Precisely how oxLDL and associated lipids instigate the atherogenic process remains intensively investigated, but an ill alliance of various pathways is emerging. Scavenger receptors, TLRs and endoplasmic reticulum stress in macrophages. Although SR-A and CD36 mediate the majority of modified LDL uptake by macrophages in vitro, their genetic deletion has yielded varying effects on mouse atherosclerosis, possibly reflecting differences in the genetic background, regional context or inflammatory features studied76. Combined deficiency of SR-A and CD36 in Apoe/ mice has implicated these receptors in atheroprogression, by inducing proinflammatory gene expression and macrophage apoptosis, rather than foam cell formation77. TLR4 and TLR6 cooperate with CD36 in this inflammatory response of macrophages to atherogenic lipids; that is, oxLDL sequestered by CD36 induces intracellular CD36-TLR4-TLR6 heteromerization, activating NF-B and chemokine expression78 and explaining the proatherogenic effects of TLR4 (ref. 32). Accordingly, a sustained oxidative burst and apoptosis elicited by atherogenic lipids in macrophages with continuous endoplasmic reticulum (ER) stress33 through combined signaling of CD36 with TLR2-TLR6 may similarly occur in human or mouse lesional macrophages with intracellular accumulation of free cholesterol41 (Fig. 4).
Figure 4 Lipidmediatorsaffect LDL inflammationandatherogenesisthrough Monocyte Monocyte adhesion diversesignalingpathways.OxLDLbindsCD36, CXCL1 inducingintracellularCD36- TLR4-TLR6heterotrimerization,which LPA1/3 EC activatesNF-Bandchemokineexpression Lp-PLA2 oxLDL, ox-phospholipids Unsaturated LPA 78.Atherogenic LPC LPA bylesionalmacrophages lipoprotein(a), fatty acids + TLR4/6 lipidmediators,suchasoxLDL,oxidized oxLDL oxfatty acids TLR2/6 phospholipidsandlipoproteinsandfatty acids,alsotriggerNADPHoxidase Modified receptor CD36 ERK activationthroughaCD36-TLR2-TLR6 presentation and signaling NADPH pathway,resultinginasustainedoxidative oxidase Apoptosis ROS burstandapoptosisofER-stressed Lysosome Cholesterol-rich MyD88 cholesterol-overloadedfoamcells33. lipid raft TRIF Furthermore,anintracellularexcessoffree Cholesterol Free ER NF-B cholesterol,forexampleduetodefective efflux cholesterol stress ABCG1 cholesteroleffluxbyABCtransporters, canmodifyreceptorpresentationand Caspase-1 HDL signalingbyincreasinglipidraftformation, ASC Anti-inflammatory ABCA1 whichinhematopoieticstemcellsinduces Inflammasome NLRP3 myeloproliferationduetoenhancedIL-3 activation andGM-CSF-signaling83.Also,intracellular cholesterolcrystalscanexertproatherogenic Phagolysosomal damage effectsbystimulatingIL-1productionby Proinflammatory macrophagesthroughNLRP3inflammasome Cholesterol Active IL-1 activation85,86.Lipidscanalsoaffect Chemokines crystals caspase-1 endothelialcellfunctionsthephospholipase IL-1 proIL-1 Lp-PLA2hydrolyzesoxLDL-associated phospholipidsintooxidizedfattyacids andlysophosphatidylcholine(LPC), andthederivativelysophosphatidicacid(LPA)triggersendothelialcellstosecreteandpresentCXCL1formonocyteadhesion92.Thus,oxLDLand associatedlipidsactondiversecelltypestoinstigateinflammationandproatherogenicprocesses.CASP1,caspase1.
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Defective efferocytosis of apoptotic cells then causes secondary necrosis. A link between ER stress, apoptosis of macrophages or other cells and necrotic core formation has been confirmed in mice with somatic deficiency in the ER stress effector CHOP79. Although antioxidant therapies in humans have failed to protect against cardiovascular disease, these findings pinpoint specific mitigation of lipotoxic ER stress and the resulting oxidative burst as potential strategies. It is therefore important to unravel the molecular mechanisms and signaling routes employed by ER stress to promote atherogenesis80. In this context, fatty acid binding protein-4 (aP2) has been identified as a regulator of macrophage ER stress responses to lipids. Blocking aP2 function mitigates lipotoxic ER stress by reactivating de novo lipogenesis and lipid desaturation, mainly through LXR, increasing bioactive lipid production and reducing macrophage apoptosis and atherogenesis81. Reverse cholesterol transport and myeloproliferation. Besides inducing ER stress in macrophages, intracellular free cholesterol can also integrate into cellular membrane lipid rafts. Defective cholesterol efflux due to deficiency of the ABC transporters ABCA1 and ABCG1 (ref. 82) can enhance cholesterol-rich raft formation in hematopoietic stem cells, increasing surface expression of the common IL-3 and GM-CSF receptor -subunit, which, in turn, increases IL-3 and GM-CSF triggered downstream signaling and myeloproliferation83 (Fig. 4). Mice with ABCA1- and ABCG1-deficient bone marrow showed accelerated atherosclerosis82, whereas myeloproliferation and atherosclerosis were reversed by promoting cellular cholesterol efflux to HDL and in ApoA-1 transgenic mice with increased HDL levels83. The inverse relationship between HDL and leukocytosis/atherogenesis confirms human correlation studies5, identifies suppression of myeloproliferation as an HDL-mediated anti-atherogenic mechanism and points to HDL as an attractive therapeutic beyond its cholesterol effluxpromoting capacity. The discovery of molecules promoting cholesterol efflux could extend such options. For example, defective sphingosine-1-phosphate receptor signaling in macrophagesshifting the balance from oxLDL uptake to reverse cholesterol transportdecreases atherogenesis84. Inflammasome activation by cholesterol crystals. An intriguing finding is the presence of small cholesterol crystals in human and mouse atherosclerotic lesions85,86. The crystals appear simultaneously with lesional macrophages, accumulate in subendothelial and necrotic areas and promote atherogenesis by activating the NLRP3 inflammasome, a PRR platform mediating IL-1 secretion (via caspase-1) and neutrophil recruitment85,86 (Fig. 4). Unlike Ldlr/ mice with bone marrow deficiency of NLRP3, ASC or IL-1/, Apoe/ mice lacking NLRP3, ASC or caspase-1 were not protected against lesion progression or infiltration87. This discrepancy may be due to genetic background differences, irradiation, protective functions in nonbone marrowderived cells, or minor contributions of the NLRP3 inflammasome and a more prominent calpain-mediated IL-1 generation in hyperlipidemic Apoe/ mice. Thus, interference with cholesterol crystal formation or inflammasome activation may be a therapeutic strategy for preventing atherogenesis under defined conditions. Whether cholesterol crystals precede monocyte recruitment or accompany macrophages as intracellular passengers, as well as the molecular mechanisms underlying inflammasome activity in human disease, remain to be elucidated. Lp-PLA2 and lysophosphatidic acid. The lipoprotein-associated phospholipase A2 (Lp-PLA2) primarily acts on oxLDL to generate
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proinflammatory mediators such as lysophosphatidylcholine (LPC) and oxidized nonesterified fatty acids. Lp-PLA2 is highly expressed in human vulnerable atherosclerotic lesions, and increased Lp-PLA2 activity correlates with proatherogenic lipids and cardiovascular risk in humans88. Selective inhibition of Lp-PLA2 with darapladib reduces development of complex CAD in swine, limiting LPC content, lesions with an unstable phenotype and inflammatory gene expression89. Recent evidence showing that oxidized phospholipids are primarily cleared through transport rather than Lp-PLA2 (ref. 90) indicates that adverse effects of Lp-PLA2 inhibition by affecting oxidized phospholipid degradation are unlikely. Likewise, the LPC derivative lysophosphatidic acid (LPA) has been implicated in atherogenesis, given the increased LPA levels in plaques and serum upon hypercholesterolemia and its involvement in activation of and interactions between endothelial cells, monocytes and platelets91. Notably, unsaturated LPA promotes macrophage accumulation and lesion progression in atherosclerosis-prone mice, whereas combined LPAR1 and LPAR3 receptor blockade reduces foam cell content and atherogenesis 92. The effects on macrophage populations could be explained by binding of unsaturated LPA to endothelial LPAR1 and LPAR3, inducing secretion and surface immobilization of CXCL1 to mediate arterial monocyte adhesion92 (Fig. 4). In conclusion, lipid mediators, including phospholipids, cholesterol, fatty acids and lipoproteins, affect inflammation and atherogenesis through diverse pathways. Unraveling these mechanisms at the molecular level could catalyze the development of innovative cardiovascular disease therapeutics. The identification of new players involved in lipid metabolism through large-scale genome-wide association studies (GWASs) of lipid- and lipoprotein-levelcorrelated genetic variants93 should accelerate this process. Genetic and metabolic profiling studies To complement traditional risk factors, such as hyperlipidemia and hypertension, a search for genetic variants linked with cardiovascular disease has flourished, and GWASs have created an enormous wealth of data on susceptibility loci (Table 1)9499. But not all associations reported have been independently replicated, and it remains unclear whether multilocus cardiovascular disease genetic risk scores based on GWAS-identified chromosomal loci have predictive abilities better than traditional risk factors96. Nonetheless, characterization of cardiovascular diseaseassociated loci99 should provide new insight in the molecular mechanisms driving atherosclerosis and cardiovascular disease and open new avenues for cardiovascular drug research. New risk alleles for CAD. The strongest genetic effect on CAD and myocardial infarction is carried by locus 9p21.3 (ref. 94,97), which contains several coding genes (CDKN2A, CDKN2B and MTAP) and an antisense noncoding RNA (CDKN2B-AS, also known as ANRIL) that alters gene regulation94. The risk alleles of the single nucleotide polymorphisms (SNPs) rs10811656 and rs10757278 were found to disrupt a STAT1 binding site and affect signaling by IFN- 99, a complex regulator of atherosclerosis. Another example is locus 10q11.21, wherein two SNPs rs1746048(C/C) and rs501120(T/T) have been associated with increased CAD and myocardial infarction risk94,95,97. The risk alleles of these SNPs, approximately 80 kb downstream of the chemokine gene CXCL12, correlated with increased CXCL12 plasma levels, suggesting CXCL12 as a proatherogenic molecule100. But other studies suggest an atheroprotective function for CXCL12 on the basis of the association of reduced CXCL12 plasma levels with unstable angina101 and CAD risk allele rs501120T/T102. Thus, the precise
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Table 1 Selection of CAD- and myocardial infarction (MI)-associated loci identified by GWAS
Locus 1p13.3 Candidategene(s) Locatedwithinoradjacent tothislocus SORT1 SNP rs599839 rs646776 rs11206510 rs3008621 rs17465637 rs6725887 rs9818870 rs2306374 rs12526453 rs9349379 rs3798220 rs2048327,rs3127599 rs7767084,rs10755578 rs1333049 rs10738610 rs4977574 rs10757278 10q11.21 12q24 12q24.12 12q24.3 19p13.2 21q22.11 CXCL12 SH2B3 SH2B3 HNF1A,C12orf43 LDLR SLC5A3,MRPS6,KCNE2 rs501120 rs1746048 rs11065987 rs3184504 rs2259816 rs1122608 rs9982601 Riskallele A T T G C C T C C C C CTTG,CCTC C C G G T C G T T G T Riskallelefrequency (%)(European Disease ancestry) association 7778 7981 8184 72 7275 1415 1015 18 6567 44 12 18 52 48 4356 46 84 8487 34 40 36 7579 1315 CAD+MI CAD+MI MI CAD+MI MI CAD CAD CAD CAD+MI CAD CAD+MI CAD+MI CAD+MI CAD+MI CAD+MI CAD+MI CAD+MI CAD+MI CAD MI CAD CAD+MI CAD+MI SNPeffect IncreasedLDL cholesterollevel References 94,97 95,98a 95a 94,96 95,97a 94,97a 94a 97 94,95,97 96 96,97 94 94 94 9597 99 94 95,97a 94 94,96 94a 9496 9497
1p32.3 1q41 2q33.1 3q22.3 6p24.1 6q25.3 6q26-27 9p21.3b
PCSK9 MIA3 WDR12,ALSC2R13 MRAS PHACTR1 LPA SLC22A3,LPAL2,LPA MTAP,CDKN2A,CDKN2B, CDKN2B-AS (or ANRIL)
IncreasedLp(a)level
DisruptedSTAT1 bindingsite
aSignificantassociationwithCADorMIwasnotconfirmedinreference96.bStrongestassociationwithCADandMIofallloci.
role of CXCL12 and its receptor CXCR4 in atherosclerosis remains unclear and will require large-scale CAD and myocardial infarction meta-analysis studies combined with animal and molecular studies for elucidation. New atheroprotective mechanisms. Interference with the CXCR4CXCL12 axis by systemically treating mice with a CXCR4 antagonist or transplanting them with CXCR4-deficient or CXCR4-degrakine transduced bone marrow aggravates diet-induced atherosclerosis as a result of severe leukocytosis and neutrophilia35. Furthermore, microRNA-126 (miR-126) or miR-126carrying endothelial apoptotic bodies have been identified as atheroprotective in mice, promoting CXCR4-dependent mobilization and lesional incorporation of progenitor cells. miR-126 mediates degradation of RGS16, which unleashes autoregulatory CXCR4 signaling and increases endothelial production of CXCL12, a known mediator of progenitor cell mobilization103. Thus, local CXCL12 induction conferred by miR-126 in endothelial apoptotic bodies may pomote atheroprotection, which can be enhanced by miR-126mediated repression of endothelial vascular cell adhesion molecule-1 expression104, limiting inflammatory cell arrest. Recent miRNA profiling studies discovered that circulating levels of vascular-derived miRNAs, including miR-126, were reduced in individuals with CAD105, suggesting that miR-126s atheroprotective functions may be exploited by therapeutic mimetics or miR-126loaded microparticles. The CXCR4- and CXCL12related roles of neutrophil homeostasis, and endothelial homeostasis, as well as the involvement of miR-126 and its targets in atheroprotection emphasize the need to address these axes in cell-type specific conditional knockouts.
Gut flora and metabolically driven atherogenesis. The intestinal microbiotic flora performs essential functions in nutrient processing and assists host immune responses, but it has also been linked to metabolic and immune disorders, prompting studies of its relation to human disease. A recent metabolomics approach identified the dietary lipid phosphatidylcholine and its metabolites choline, betaine and trimethylamine N-oxide (TMAO) as risk factors for cardiovascular disease. In mice, a choline-rich diet increased TMAO levels and atherosclerosis, depending on gut flora activity, as shown by broad-spectrum antibiotics treatment106. Although earlier antibiotic trials failed to reduce event rates in humans with CAD107, specific targeting of bacterial species responsible for choline metabolism using selective antibiotics or probiotics might have therapeutic value for cardiovascular disease106. Detailed analysis of the relations between gut microbiota, host metabolism and atherosclerosis may prompt subtle microbiota manipulation as a new way to modulate lipid metabolism and treat CAD. Atherosclerosis therapy The high incidence of atherosclerosis-related cardiovascular disease imposes an enormous burden on healthcare systems. Currently available therapeutics against atherosclerosis are largely restricted to alleviating hypertension and hyperlipidemia or controlling hemostasis to prevent thrombotic complications. But these strategies do not directly address the inflammatory mechanisms driving atheroprogression. Here we survey established, emerging and future concepts in the treatment of atherosclerosis (Table 2). Established therapies. In patients with CAD, statin treatment for secondary prevention yields a pronounced risk reduction, which
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Table 2 Established, emerging and new experimental concepts in the treatment of cardiovascular disease
Compoundormethod Established therapies Statins,forexample,atorvastatinand rosuvastatin Nicotinicacid(niacin) Aspirin,clopidogrel,prasugrel,ticagrelor -blockers Renin-angiotensinsysteminhibitors Emerging therapeutic approaches HDLmimetics,forexample,apoa1-Milano Darapladib(selectiveLp-PLA2inhibitor) IL-1ra(IL-1receptorantagonist) Methotrexate Pitfalls Promotecholesterolefflux,anti-inflammatory Decreasesatherogeniclipidproduction Immunosuppressive Clinicalphase1and2 Clinicalphase3 Clinicalphase2 Clinicalphase3 Adversesideeffects Anti-inflammatory Reducesappetite InhibitscholesteroltransportfromHDLtoLDL IncreasedriskofheartfailureandMI Depressiveeffectsdiscontinued Raisedsystolicbloodpressure discontinued Clinicalphase3(DEFINEstudy) Clinicalphase2 120123 124 125 117,118 89,119 126 126 Inhibitcholesterolsynthesis,anti-inflammatory Primaryandsecondaryprevention 108110 111,112 113,114 115 116 Mechanism Statusoroutcome References
Inhibitsfatbreakdowninadiposetissueandincreases Secondaryprevention HDLcholesterol,anti-inflammatory Inhibitplateletaggregation Antihypertensive Antihypertensive Secondaryprevention Secondaryprevention Secondaryprevention
Thiazolidinediones(PPARagonists) Rimonabant(cannabinoidtype-1receptor antagonist) Torcetrapib(CETPinhibitor) Possiblealternatives:anacetrapib(CETP inhibitor) Dalcetrapib(CETPinhibitor) Novel experimental strategies BlockingtheCD40-TRAF6interactionsite BlockingMIFreceptorbinding Maraviroc(CCR5antagonist) MLN1202(CCR2-specificantibody) NonagonisticCCL2-competingmutantPA508 Dominant-negativeCCL5mutant[44AANA47] Mkey(ct-2009) Immunization CCL17inhibition
BlockingCCR5 BlockingCCR2 Nonagonisticplusincreasedproteoglycan affinity Createsdimersdevoidofproteoglycanbinding DisruptsCCL5-CXCL4heteromerization Protectiveantibodygeneration,Tregcellinduction SupportsTregcellhomeostasis
Limitsatherosclerosisofunstable phenotypeinmice Induceslesionstabilizationand regressioninmice ApprovedforUSandEuropeanmarkets forHIVtreatment Clinicalphase2 Attenuateslesionformationinmice Attenuateslesionformationinmice Attenuateslesionformationinmice Canattenuatelesionformationinmice Attenuateslesionformationinmice
75 30,127,128 7 129 130 131 23 45,132135 25
correlates with lowering of LDL cholesterol. Mechanistically, statins exert a pleiotropy of anti-inflammatory actions, improve endothelial function and reduce plaque lipids and thrombogenicity, thereby limiting and stabilizing atherosclerotic plaques 108. Aggressive lipid lowering by high-dose atorvastatin decreased high-sensitivity C-reactive protein (hs-CRP) serum levels and entailed regression of atheromas, as monitored by intravascular ultrasound (IVUS)109. The clinical benefit is extended to primary prevention in patients with LDL cholesterol levels <130 mg dl 1 but elevated hs-CRP, where rosuvastatin significantly reduces major cardiovascular event rates, with reductions in both LDL cholesterol (<70 mg dl 1) and hsCRP indicative of successful treatment110. In ACS, aggressive statin therapy also lowered the incidence of primary endpoints including myocardial infarction, with the most substantial benefit in patients with declines in both LDL cholesterol and CRP108. In statin-treated patients with low HDL cholesterol, shifting the lipid balance in favor of HDL cholesterol by extended release of nicotinic acid (niacin) has been exploited to achieve regression of carotid intima-media thickness, although this is probably owing to anti-inflammatory
effects through the niacin receptor GPR109A in immune cells rather than to the lipid-modifying effects of niacin111,112. Platelet inhibition with aspirin has proved an enduring pillar for secondary prevention in patients with ACS, a benefit enhanced by other antiplatelet agents, such as clopidogrel, prasugrel or ticagrelor113. However, its limited suitability for primary prevention of atheroprogression is probably related to the atherogenic effects of the platelet secretome, which remain unaltered, as aspirin primarily interferes with aggregation114. Furthermore, IVUS trials suggest that antihypertensive beta blockers lower mortality from myocardial infarction and delay atheroprogression115. Likewise, interference with the renin-angiotensin system improves endothelial function and reduces coronary event rates disproportionately to lower blood pressure, supporting direct atheroprotective effects116. Emerging therapeutic approaches. Artificial HDL-like apolipoprotein A1 complexes (apoA1-Milano) hold promise in mediating regression of CAD117. Some peptide-based HDL mimetics promote cholesterol efflux by avidly binding oxidized lipids, exert anti-inflammatory
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properties, and, based on early human studies with orally available forms, may harbor additive benefit in statin-treated patients118. After encouraging results in swine89, the selective Lp-PLA2 inhibitor darapladib failed to decrease plaque deformability but prevented expansion of the necrotic core size that reflects plaque vulnerability in patients with CAD119. A potential benefit of darapladib on coronary atherosclerosis should be established in a phase 3 trial that is currently under way. Other examples illustrate inherent pitfalls of cardiovascular drug development. Although plaque-stabilizing effects of the antidiabetic PPAR agonists thiazolidinediones have been reported in nondiabetic individuals120, conflicting evidence has warranted restrictions in their use. Addition of rosiglitazone to glucose-lowering therapy failed to reduce cardiovascular events in patients with type 2 diabetes and, even worse, raised the risk of heart failure121, and a meta-analysis of randomized trials further revealed an increased risk for myocardial infarction122. Likewise, pioglitazone, despite lowering cardiovascular risk in patients with diabetes, has been linked to serious heart failure123. Off-target effects or ambiguous results have discouraged other developments. The selective cannabinoid type-1 receptor antagonist rimonabant failed to delay progression of CAD in obese individuals with metabolic syndrome and caused psychiatric adverse effects124. Although the cholesteryl ester transport protein (CETP) inhibitor torcetrapib induced regression of coronary atherosclerosis at the highest HDL-cholesterol levels achieved, it also showed aldosteronerelated adverse effects125. This may explain a lack of benefit in the full study cohort but still suggests that CETP inhibitors without offtarget activity, for example, anacetrapib or dalcetrapib, may halt plaque progression. In addition, better insights into the relation between HDL-particle content and beneficial function are necessary. These examples underscore a need for ongoing identification and validation of alternative targets and drug candidates. Studies using currently available anti-inflammatory treatment regimens for atherothrombosis, such as IL-1 receptor antagonism or immunosuppressive methotrexate, are currently under way126. New experimental strategies. The characterization of inflammatory and immune reactions underlying atherogenesis has uncovered new therapeutic strategies. Specific disruption of the CD40-TRAF6 interaction site without disturbing other CD40 signaling cascades limits atherosclerosis of an unstable phenotype by skewing the immune response toward a protective profile and may circumvent thromboembolic complications associated with CD40L-specific antibody administration in humans73,75. Similarly, blocking MIF receptor binding to induce lesion stabilization and regression30 could be explored with peptide antagonists to the pseudo-(E)LR and N-looplike motif of MIF for CXCR2 binding30,127,128. However, a potential application of MIF inhibitors as human therapeutics requires careful evaluation, as adverse effects may arise from blocking its other functions, including its redox and tautomerase activities71. Related to MIF, interference with chemokines has gained attention in cardiovascular drug research (Fig. 1). The quest for HIV entry inhibitors has led to approval of maraviroc, an antagonist of the proatherogenic chemokine receptor CCR5. As the safety of this drug is sufficiently documented, it seems worthwhile to assess its effects on plaque progression and stability in patients with CAD7. Direct blockade of CCR2 using the antibody MLN1202 reduced CRP levels in patients at risk for cardiovascular disease in a phase 2 trial129 and may be suitable to supplement statin therapy. Targeting oligomerization and proteoglycan binding of chemokines may extend therapeutic options: the nonagonistic CCL2-competing
mutant PA508, which has increased proteoglycan affinity, and the dominant-negative CCL5 mutant [44AANA47], which creates dimers devoid of proteoglycan binding, attenuated lesion formation in mice7,130,131. As chemokines are an integral part of the immune system, blocking these constituents carries a risk of unwanted side effects. Tailored manipulation of heterophilic chemokine interactions introduces a promising alternative that leaves normal physiological, immunological functions intact. Disrupting CCL5-CXCL4 heterodimerization with the cyclized peptide MKEY selectively inhibited heteromer-mediated enhancement of atherogenic monocyte recruitment and atherosclerosis in hyperlipidemic mice without affecting other CCL5-related functions or compromising immune responses and host defense23. Assessing toxicological screening and validating the effectiveness of these drugs will require vigorous testing in clinical settings. Exploiting the chemokine interactome for context-specific interference may apply to other predicted atherogenic interactions7. Using adaptive immunity to control immune responses sustaining atherogenic inflammationimmunization with LDL, oxLDL, heat shock proteins or ApoB100 fragments can reduce atherosclerosis in hyperlipidemic animalsimplicates vaccination as an approach to prevent or mitigate disease49. Whereas the best antigen and study population for vaccination remains unclear, antigen-pulsed DCs have been explored as an adjuvant strategy. Repetitive injection with lipopolysaccharide-matured DCs pulsed with SMC-derived artificial self antigen or aldehyde-modified LDL aggravated atherosclerosis during high-fat diet45,132. In contrast, injecting lipopolysaccharidematured DCs pulsed with oxLDL before high-fat diet favored more stable plaque formation133, and single administration of tolerogenic IL-10exposed, ApoB100-pulsed DCs before high-fat diet attenuated atherosclerosis in human ApoB100-transgenic mice134. In addition, mucosal immunization with an ApoB100 peptide was associated with antigen-specific Treg cell generation and reduced cellular immunity to ApoB100 (refs. 134,135). These conflicting results can be reconciled by the concept that the degree of hyperlipidemia, timing of application and local tolerogenic milieu may shape functional phenotypes of endogenous or transferred DCs. In this context, short-circuiting the restraining effect of mature CCL17+ DCs on Treg cell homeostasis through CCL17 inhibition25 may help to create a regulatory IL-10biased environment to improve tolerogenic DC therapy or disrupt DC-orchestrated vicious circles. In summary, established drugs, such as niacin, owing to improved formulations or combination with a prostaglandin D1 receptor antagonist to reduce flushing, could regain attention, whereas emerging compounds such as CETP or Lp-PLA 2 inhibitors could be further refined and tested in large-scale trials4 with a focus on excluding deleterious off-target effects. New strategies, including peptides targeting the chemokine interactome and vaccination, are emerging, but they will have to overcome translational challenges, such as specificity and efficacy. Clearly, more animal studies of established and unstable atherosclerosis are warranted for extrapolation to human disease before evaluation of new drug candidates in clinical trials. Conclusions and perspectives The response-to-injury hypothesis of Russell Ross introduced that monocyte-endothelial interactions giving rise to foam cells and growth factorinduced SMC proliferation trigger lesion formation. Pervasion of the atherogenic process has been considerably refined by the appreciation of subendothelial ApoB-lipoprotein retention, the role of macrophage-, neutrophil-, T cell and DC-driven pathways,
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and the identification of signals, such as chemotactic cytokines, regulating the lesional recruitment and homeostasis of inflammatory cells. Close links between lipid and inflammation biology continue to emerge as the pathogenic interface, as illustrated by the role of ABC transporters and ApoE in hematopoietic cell proliferation, the cholesterol crystalinduced inflammasome activation or the additional unexpected players unearthed by the gold-mining efforts of genome-wide and metabolic profiling studies. As for the chemokine interactome, these findings could offer an abundance of highly specific targeting options, which may further benefit from nanoparticlebased systems for imaging-guided and local delivery. Drug development to treat atherosclerosis requires stringent evaluation of effectiveness, whichgiven the enormous toll of clinical endpoint studies beyond lipid loweringhas to rely on surrogate markers. Although a variety of soluble, imaging or functional biomarkers is available, their predictability in phase 2 for atherosclerosis outcomes in phase 3 studies remains limited. Hence, new biomarkers should be developed to complement existing ones, or multiple biomarkers should be combined in an integrated approach. Associated drawbacks notwithstanding, GWASs and transcriptional, proteomic and metabolic profiling may facilitate this search, with the ultimate goal to model multimarker CAD scores with a higher predictive ability than established biomarkers. There is an urgent need for improved imaging techniques, as current modalities (IVUS, carotid intima-media thickness, quantitative coronary angiography, computed tomography) provide only quantitative and partial morphological data of atherosclerotic lesions, and they are often invasive and associated with ionizing irradiation. However, as cellular and molecular composition better reflects lesion pathology compared to size, highly advanced imaging techniques, namely high-resolution magnetic resonance imaging and positronemission tomography, will be required. The development of new molecular imaging probes with specific biological functions could add new dimensions to plaque imaging, including monitoring the activation of endothelial cells and macrophages. If successful, this will be of tremendous value for drug development and testing efficacy, allowing for detailed mechanistic insights into therapeutic effects at early stages, as well as for imaging-based clinical outcome studies.
Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft (FOR809), the European Research Council and Fondation Leducq. We sincerely apologize to all scientists whose important contributions to the field could not be cited due to space limitations. comPetIng FInAncIAl InteRests The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.
Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.
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Atherosclerosis: current pathogenesis and therapeutic options

2011 Nature America, Inc. All rights reserved.

VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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nature medicine VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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VOLUME 17 | NUMBER 11 | NOVEMBER 2011

NET Thrombus formation Neutrophil activation

Monocyte high (Ly6C )

HMGB1 DNA ssDNA RNA

Cathepsin G Nucleosomes neutrophil elastase

Eat me Resolution signals Anti-inflammatory Scavenging

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nature medicine VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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nature medicine VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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VOLUME 17 | NUMBER 11 | NOVEMBER 2011

1p32.3 1q41 2q33.1 3q22.3 6p24.1 6q25.3 6q26-27 9p21.3b

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nature medicine VOLUME 17 | NUMBER 11 | NOVEMBER 2011

Inhibitsfatbreakdowninadiposetissueandincreases Secondaryprevention HDLcholesterol,anti-inflammatory Inhibitplateletaggregation Antihypertensive Antihypertensive Secondaryprevention Secondaryprevention Secondaryprevention

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BlockingCCR5 BlockingCCR2 Nonagonisticplusincreasedproteoglycan affinity Createsdimersdevoidofproteoglycanbinding DisruptsCCL5-CXCL4heteromerization Protectiveantibodygeneration,Tregcellinduction SupportsTregcellhomeostasis

75 30,127,128 7 129 130 131 23 45,132135 25

VOLUME 17 | NUMBER 11 | NOVEMBER 2011

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12. 13. 14. 15.

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31. 32. 33.

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