Atherosclerosis and related ischemic diseases: inflammation, the mixed blessing





Jean-Sébastien SILVESTRE, PhD



Alain TEDGUI, PhD
Paris Cardiovascular Research Center, Inserm UMRS 970
Université Paris Descartes
Sorbonne Paris Cité
Paris, FRANCE

Atherosclerosis and related ischemic diseases: inflammation, the mixed blessing

by J. S. Silvestre and A. Tedgui, France

Inflammation plays a major role at all stages of the atherosclerotic process, from the early events, whereby leukocytes are recruited at sites of subendothelial LDL cholesterol accumulation, to the late events, when plaque rupture occurs and leads to thrombus formation. Clinical complications of atherosclerosis, such as myocardial infarction or critical limb ischemia, are also featured by the immunoinflammatory reaction within the affected territory that plays an active role in postischemic vascular and tissue remodeling. A complex continuum of molecular, cellular, and extracellular responses is controlled by the different actors of the inflammatory reaction and determines the extent of atherosclerosis development, as well as the homeostasis of the ischemic tissue. Although therapeutic targeting of inflammatory cells and/or cytokines may be envisioned for a short period of time following acute coronary events, such a therapeutic approach should target a specific type (or subtype) of inflammatory cells and should deeply evaluate the long-term effects of inflammatory mediators on atherosclerosis and ischemic tissue regeneration. This review provides an overview of our current knowledge regarding the role of innate and adaptive immunity in atherosclerosis and related ischemic diseases.

Medicographia. 2014;36:355-361 (see French abstract on page 361)


Inflammation and atherosclerosis

Atherosclerotic plaques develop in predisposed areas of the arterial tree where blood flow is either slow or oscillatory. In these predisposed areas, endothelium displays increased susceptibility to activation as well as greater permeability to low density lipoproteins (LDL), favoring subendothelial retention in these locations. One of the triggering events in the initiation of atherosclerotic lesion formation is the oxidation of LDL, which is mediated by both enzymatic and nonenzymatic mechanisms. The infiltration, retention, and oxidation of LDL promote the release of bioactive lipids in the arterial intima, which activates vascular cells to produce proinflammatory and chemotactic factors.1 These molecules attract lymphocytes and monocytes. In the arterial intima, the immune cells generate various cytokines and chemokines, which leads to a cascade of events, involving recruitment of more inflammatory cells, further lipid accumulation, and increased migration, differentiation, and proliferation of smooth muscle cells (SMCs). Infiltrated monocytes differentiate into macrophages or dendritic cells. Resident subendothelial dendritic cells are believed to be the first cells to take up oxidized LDL (oxLDL) to become foam cells.2 However, most of the foam cells arise from macrophages that internalize oxLDLthrough scavenger receptors, leading to the accumulation of cholesterol esters. Surprisingly, early acquisition of cholesterol by macrophages suppresses inflammatory responses, and promotes a reparative macrophage phenotype.3 Yet, continued cholesterol accumulation results in predominantly inflammatory macrophages. Macrophages and other immune cells that accumulate under hypoxic conditions undergo apoptosis and necrosis, leading to the formation of the necrotic core and an unstable plaque. Advanced, rupture-prone lesions that are associated with clinical events contain increased numbers of macrophages and T cells, large necrotic cores, but thinned fibrous caps with fewer SMCs and collagen content. The chronic inflammatory disease of the arterial wall is promoted by both innate and adaptive immunity.4

Innate immunity
The innate response is instigated by the activation of vascular cells and monocytes/macrophages. Subsequently, an adaptive immune response develops against an array of potential antigens presented to effector T lymphocytes by antigen- presenting cells. Experimental studies in murine models of atherosclerosis have shown that proinflammatory and T helper 1 (TH1)–related cytokines promote the development and progression of the disease, whereas anti-inflammatory and regulatory T cell–related cytokines exert clear antiatherogenic activities.5 As an example, signaling through the interleukin- 1 receptor (IL1R) severely aggravates vascular inflammation and atherosclerosis.6 IL1R-deficient (IL1R1−/−) mice exhibit less atherosclerosis, and overexpression of the IL1R antagonist (IL1RA) ameliorates the disease. Conversely, mice deficient in IL1RA (IL1RN−/−) display exacerbated atherosclerosis, and spontaneous and fatal vascular inflammation. In the early stages of atherosclerosis, proinflammatory cytokines can alter endothelial functions. Tumour necrosis factor α(TNFα), for example, increases cytosolic Ca2+ and activates myosin light chain kinase and ras homolog gene family, member A (RhoA), which disrupts endothelial junctions, leading to loss of barrier function and facilitation of leukocyte transmigration. Cytokines also induce the expression of chemokines and adhesion molecules in endothelial cells, favoring the recruitment, adhesion, and migration of lymphocytes and monocytes into the inflamed vessel wall. Once in the intima, leukocytes can be permanently activated by locally generated cytokines, which can accelerate the transformation of macrophages into foam cells by stimulating the expression of scavenger receptors and enhancing cell-mediated oxidation. Cholesterol crystals act as metabolic triggers of the NLRP3 inflammasome, which promotes the maturation of IL-1β and IL-18.7 At an advanced stage of the disease, proinflammatory cytokines destabilize atherosclerotic plaques by promoting cell apoptosis and matrix degradation. A number of proinflammatory cytokines can induce SMC and macrophage apoptosis, particularly the association of IL-1, TNFα, and interferon ϒ(IFN-ϒ). Macrophage apoptosis results in the formation of cell debris, which contributes to the enlargement of the lipid core.Plaque SMC apoptosis leads to thinning in the fibrous cap, favoring its rupture. Proinflammatory cytokines significantly affect the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), inducing substantial remodeling of many components of the extracellular matrix. For example, IFN-ϒ inhibits collagen synthesis, whereas IL-1 and TNFα induce a broad range of MMPs in vascular cells, including MMP-1, 3, 8, and 9. Finally, the antithrombotic properties of endothelial cells are deeply altered by cytokines. Downregulation of anticoagulant mediators may in turn affect inflammation. Proinflammatory cytokines also modify the fibrinolytic properties of endothelial cells, decreasing the production of tissue plasminogen activator and increasing the production of type I plasminogen activator inhibitor. As a result, proinflammatory cytokines might precipitate thrombus formation and promote the development of acute coronary syndrome (ACS).





A variety of plasma inflammatory markers have been shown to predict future cardiovascular risk well. They can be useful for risk stratification and also to identify those patients who might benefit from targeted interventional therapy.1 Of these markers, C-reactive protein (CRP), an acute-phase protein, has been the most extensively studied. There is robust evidence from primary prevention cohorts and amongst patients presenting with ACS that elevated CRP levels predict future cardiovascular events. Also, IL-6 levels appear to be predictive of cardiovascular events and are elevated in patients with unstable angina compared with those with stable angina. In a recent meta-analysis including up to 133 449 individuals, an IL-6 receptor (IL6R) single nucleotide polymorphism (rs7529229) was associated with increased circulating IL-6 concentration, consistent with IL6R blockade, and decreased coronary heart disease events, suggesting that IL6R signaling has a causal role in the development of coronary artery disease.8

Hitherto, the strongest evidence that inflammation plays a major role in atherosclerosis in humans stems from studies in patients with autoimmune disease who are at very high cardiovascular risk, and who benefit from anti-inflammatory treatments. Patients with autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis, are at particularly high risk of cardiovascular disease. In patients with rheumatoid arthritis, anti-TNF therapy reduced inflammation, thrombotic risk, and the incidence of cardiovascular events.9

♦ Adaptive immunity
T-cell responses are initiated when specific molecular epitopes on antigens, including oxLDL and heat shock proteins, are presented by antigen-presenting cells and recognized by T cell antigen receptors. Although macrophages can also present antigens to T cells, dendritic cells are the main cell type responsible for the activation of naive T cells and therefore play a crucial role in triggering adaptive immunity. In atherosclerotic plaques, dendritic cells colocalize with T cells, suggesting that they are involved in T-cell activation within the plaque. However, sensitization of naive T cells most likely occurs in the regional lymph nodes.5 A number of experimental studies have clearly shown a critical pathogenic role for the TH1 response, associated with the production of IFN-ϒ.5


Figure 1
Figure 1. Immunoinflammatory balance and atherosclerosis.

Lymphoid organs are specialized in antigen presentation and may be the major site of pathogenic
or tolerogenic antigen presentation and T-cell priming in atherosclerosis. Antigen presentation
may also occur within the atherosclerotic plaque, which is rich in cells with antigen
presenting capacity (macrophages and dendritic cells). Continuous trafficking of immune cells
between the inflamed atherosclerotic artery and the lymphoid organs may be necessary to
mount an adaptive immune response. CD28 engagement and IL-2 production are required
for regulatory T lymphocyte (Treg) survival and maintenance in the periphery. Treg cells suppress
the pathogenic response through IL-10, TGF-β. and/or cell-cell contact-dependent mechanisms.
Abbreviations: APC, antigen-presenting cell; IFN, interferon; IL, interleukin; Mac, macrophage;
TGF, tumour growth factor; TH, T helper cell.



Atherosclerosis is also associated with B-cell activation. The main function of B cells is to secrete antibodies of various isotypes. Immunoglubulin G (IgG) antibody production by B2 B cells, the most common type of B cells, requires T cell co-stimulation, whereas innate production of natural immunoglobulin M (IgM) antibodies by B1 B cells does not require the help of T cells. Both IgG and IgM antibodies against oxLDL have been described. In mouse models, recent studies have provided evidence for a proatherogenic role of B2 B cells.10

Natural regulatory T lymphocyte (Treg) cells develop in the thymus and recognize a specific self-antigen. They are characterized by the expression of CD4, high levels of CD25, and the transcription factor Foxp3. They home to peripheral tissues to maintain self-tolerance and prevent autoimmunity by inhibiting pathogenic lymphocytes. These cells mediate suppressor function through the production of IL-10 and transforming growth factor β (TGFβ) Treg cells are detected in much lower amounts in atherosclerotic plaques than in other chronically inflamed tissues, such as in the skin of patients with eczema or psoriasis, where Treg cells can represent up to 25% of all Tcells, suggesting an impairment of local tolerance against potential antigens in atherosclerotic lesions.11 Using mice with genetically altered Treg cells, or by application of CD25-neutralizing antibodies, it has been shown that Treg cells exert a protective role in atherosclerosis (Figure 1).12 The effects of Treg cells are dependent on TGFβ and IL-10. In humans, a decrease in the percentage of these cells has been observed in patients with ACS.13

Therapeutic perspectives
Inflammation plays a major role at all stages of the atherosclerotic process, from the early events, whereby leukocytes are recruited at sites of subendothelial LDL cholesterol accumulation, to the late events, when plaque rupture occurs, leading to thrombus formation and adverse clinical outcomes. The chronic inflammatory disease of the arterial wall is promoted by both innate and adaptive TH1-driven immunity, and is orchestrated by a complex network of proinflammatory cytokines. Murine experimental models of atherosclerosis provide clear evidence that blockade of proinflammatory cytokines results in limitation of plaque development and progression. In humans, anticytokine therapies have proven very successful against autoimmune disease. However, most of the proinflammatory cytokines are central to a successful host defense against microbial pathogens. Therefore, although therapeutic targeting of these cytokines may be envisioned for a short period of time following acute coronary events, it is unlikely to be accepted for long-term treatment of atherosclerosis, given the cost/benefit ratio. As more is discovered about the complex role of adaptive immunity, especially the atheroprotective effects of Treg cells mediated by IL-10 or TGFβ, more subtle therapeutic approaches, adapted to specific long-term treatment and aimed at limiting lesion development and atherosclerosis-related inflammation will be developed.


Figure 2
Figure 2. Recruitment of inflammatory cells to atherosclerotic
plaque and related ischemic milieu.

The atherosclerotic process and its clinical complications, such as
myocardial infarction or critical limb ischemia, lead to the mobilization
of different subsets of inflammatory cells from the bone marrow and
the spleen to the systemic circulation. Diverse interactions between
the adhesion molecules expressed by myeloid cells and lymphocytes
(PSGL-1, CD62L, integrin β) and endothelial cells (selectin CD62E
and CD62P, VCAM-1, ICAM-1 and -2) are involved in the initial
attachment, rolling, and firm adhesion of circulating cells to the activated
endothelium. The differential kinetic of inflammatory cell subset
infiltration is mainly under the control of the chemokine families, CCchemokines
and the CXC chemokines, through interaction with their
specific receptors expressed by the different types of inflammatory cells.
Infiltrated inflammatory cells then control the outcome of atherosclerosis
and ischemic tissue through the paracrine release of cytokines,
interleukins, growth factors, reactive oxygen species, or proteases.
Abbreviations: GAG, glycoaminoglycans; GPCR, G protein coupled
receptor; Ly6Chigh, Ly6C high monocytes; Ly6Clow, Ly6C low monocytes;
T cell, T lymphocytes; TGF, tumour growth factor; Treg, regulatory
T lymphocyte.


Inflammation and remodeling of ischemic tissues

The pathogenesis and progression of atherosclerosis may lead to thrombotic complications, vascular occlusion, and subsequent ischemic injury of the affected tissue. Various types of inflammatory cells are also recruited to ischemic sites, where they play an active role in vascular and tissue remodeling in the context of critical leg ischemia (CLI) or myocardial infarction (MI).14

Innate immunity
Monocytes/macrophages
In an experimental model of CLI, the presence of monocytes is strongly associated with the local proliferation of endothelial cells and SMCs and the extent of the postischemic neovascularization process closely depends on the number of circulating monocytes.sup>15 Monocytes can be mobilized from the bone marrow or the spleen. In mice with MI, 40% of the monocytes infiltrating the infarcted myocardium 24 hours after the ligation originate from the spleen.16 Monocytes promote angiogenesis and collateral growth in a paracrine manner, by secreting diverse growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).17 They are also a major source of MMP-9, which is involved in the emergence and branching of the newly formed vascular network. Monocytes constitute a heterogeneous population with two major subtypes in mice: Ly6Chigh- CCR2+CX3CR1low monocytes and Ly6ChighCCR2CX3CR1low monocytes, corresponding in humans to the CD14+CD16 and CD14lowCD16+ subpopulations, respectively.18 These two subtypes of monocytes are recruited sequentially to the ischemic tissue and display different functions. They have similar capacities for the phagocytosis ofdead celldebris, but Ly6Chigh monocytes secrete large amounts of diverse proteases (MMPs, cathepsins),whereas Ly6Clow monocytes expressVEGF strongly.19 The chemokine (C-C motif) ligand 2/chemokine (C-C motif) receptor 2 (CCL2/CCR2) pathway seems to act specifically on the Ly6Chigh subpopulation of monocytes (Figure 2). Similarly, two subtypes of macrophage can be distinguished on the basis of various markers and functional criteria: M1 macrophages express the inducible nitric oxide synthase and proinflammatory cytokines, such as IL-1 and IL-12, whereas M2 macrophages produce large amounts of arginase 1, anti-inflammatory cytokine IL-10, and VEGF. It is widely agreed that this M2 population favors tissue regeneration.20

Neutrophils
The role of neutrophils in the revascularization of ischemic tissues is less well defined. The elastase secreted by neutrophils has a strong antiangiogenic effect and inhibits the proangiogenic effect of other cell types. The serine proteases produced by neutrophils (elastase and proteinase 3) induce the apoptosis of endothelial cells. Neutrophils are a major source of reactive oxygen species, an overabundance of which can damage endothelial cells, blocking the angiogenesis process. Conversely, neutrophils stimulated with granulocyte colonystimulating factor secrete VEGF and may favor angiogenesis. Neutrophils also participate in the proangiogenic effect obtained after inhibition of plasminogen activator inhibitor-1 in a mouse model of CLI.14


Figure 3
Figure 3. Immunoinflammatory balance and postischemic
tissue remodeling.

The intensity and the composition of the immunoinflammatory response control
the balance between vascular regeneration and tissue remodeling, leading to
diverse specific effects on ischemic tissue homeostasis. In most cases, phenotypic
modifications to the inflammatory cells lead to the induction of numerous
proangiogenic and proarteriogenic factors and subsequently, activation of postischemic
revascularization. However, the number, activation, and differentiation
states of each type of inflammatory cell are important, and may lead to opposite
inhibitory effects on vascular growth and remodeling. Moreover, a specific type
of immune-inflammatory response may trigger adverse tissue remodeling despite
its neovascularization-activating effect.
Abbreviations: T cells, T lymphocytes; B cells, B lymphocytes; M1 Mac, type 1
macrophages; M2 Mac, type 2 macrophages; Tregs, regulatory T cells;
Ly6Chigh, Ly6C high monocytes; Ly6Clow, Ly6C low monocytes.
Red arrow indicates inhibition and green arrow activation.



T Lymphocytes
T lymphocytes (CD4+ and CD8+) are key players in the process of postischemic neovascularization. CD4−/− or CD8−/− mice that lack CD4+ and CD8+ T cells respectively, display impaired revascularization following leg ischemia.21 The neovascularization process is affected in athymic “nude” mice, which have neither CD4+ nor CD8+ T cells. CD4+ TH17 lymphocytes have also recently been shown to have proangiogenic activity. T cells have a mostly paracrine effect on ischemic tissues, notably through the secretion of VEGF. T cells also secrete cytokines, which recruit other inflammatory cells, such as monocytes, to the ischemic zones. The prior activation of monocytes by activated T cells increases the proarteriogenic potential of these cells (Figure 3).

Adaptive immunity
Treg lymphocytes
Treg lymphocytes control the neovascularization by modulating the inflammatory response to ischemia. In mice lacking Treg cells, the number of lymphocytes and monocytes/macrophages are increased in ischemic tissues, leading to activation of the neovascularization process.22

Mast cells
Growth media conditioned with activated mast cells contain diverse proangiogenic factors (VEGF, IL-8, angiopoietin-2) and can induce angiogenesis in vitro. The mast cells recruited to ischemic sites produce VEGF. Inhibition of the mobilization of mast cells in a model of leg ischemia decreases and delays the induced neovascularization. Mast cells may also affect cardiac remodeling after MI, by modulating the phenotype of endothelial cells. Indeed, the tryptase secreted by mast cells induces the production of the chemokines CCL2 and CXCL8 in endothelial cells. Mast cell granules might be protective in MI; the injection of these granules into the ischemic heart of rats decreases cardiomyocyte apoptosis and promotes neovascularization by activating the Akt/protein kinase B–dependent pathway. Nevertheless, mast cells may also have deleterious effects on cardiac remodeling. In ischemia/reperfusion, the degranulation of resident mast cells containing active TNFα and proinflammatory cytokines can participate in the deleterious remodeling of the ischemic myocardium. The inhibition of mast cell degranulation decreases myocardial ischemia/ reperfusion lesions. Combined inhibition of the angiotensin I-converting enzyme and the mast cell chymase, resulting in the inhibition of the two principal pathways responsible for angiotensin II production, has been found to have a beneficial effect on postinfarction cardiac remodeling.14

B lymphocytes
A crucial interaction between mature B lymphocytes and monocytes has recently been unravelled. After acute MI in mice, mature B lymphocytes selectively produce chemokine (C-C motif) ligand 7 (CCL7) and induce Ly6Chigh monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Of interest, high circulating concentrations of CCL7 and B cell– activating factor (BAFF) in patients with acute MI predict increased risk of death or recurrent MI (Figure 4, page 360).23,24


Figure 4
Figure 4. Role of B lymphocytes in postischemic cardiac remodeling.

After acute myocardial infarction in mice, the release of necrotic debris by the infarcted heart activates
circulating mature B lymphocytes (1). Activated B lymphocytes selectively produce chemokine (C-C motif)
ligand 7 (CCL7) (2), and induce Ly6Chigh monocyte mobilization from the bone marrow and recruitment to
the heart through activation of chemokine (C-C motif) receptor 2 (CCR2) (3), leading to enhanced tissue
injury and deterioration of myocardial function (4). B lymphocytes are also recruited in the cardiac tissue
where they may also coordinate cardiac remodeling (5). Genetic (B cell–activating factor [BAFF]–receptor
deficiency) or antibody-mediated (CD20- or BAFF-specific antibody) depletion of mature B lymphocytes
impedes CCL7 production and monocyte mobilization, limits myocardial injury, and improves heart function.
Abbreviations: Myd88, myeloid differentiation primary response gene (88); Trif, TIR domain-containing
adapter-inducing interferon β.
Adapted from reference 24: Kim ND, Luster AD. Nat Med. 2013;19:1208-1210. © 2013, Nature Publishing
Group



Therapeutic perspectives
The overall effect of the various actors of the inflammatory component depends on the local environment. The activation and differentiation states of each cell type of inflammatory cells are also important. Macrophages and differentiated dendritic cells are, for example, less able to promote vascular neogenesis than monocytes.15,25 In addition, proinflammatory cytokines, such as IL-18 and IL-12, have antiangiogenic activities.26 Antiangiogenic molecules are produced during inflammatory reactions and participate in the tissue response to ischemia.27,28 The effect of inflammation on tissue remodeling may depend on the homeostasis of ischemic tissues. Hence, results obtained in models of moderate tissue ischemia, such as experimentally induced hindlimb ischemia, may differ from those obtained after experimental models of MI, which are associated with severe tissue ischemia and massive cellular death. Moreover, the type of immunoinflammatory response may trigger adverse tissue remodeling despite its neovascularization- activating effect. Lymphocytes, like myeloid cells, display activities potentially deleterious in tissue remodeling, particularly after an MI. Excessive inflammation is associated with a decrease in left ventricle ejection fraction in mice with MI.29 T lymphocytes of patients with MI are hyperactivated and produce the proapoptotic protein FasL (Fas/CD95/ Apo-1 receptor ligand). A protective role of Treg cells has been shown in models of brain ischemia and MI. Similarly, despite their angiogenic activity, certain proinflammatory interleukins favor an excessive immune reaction in infarcted myocardium and are involved in deleterious remodeling (fibrosis) of the left ventricle. For example, the inhibition of IL-6 by neutralizing antibodies decreases inflammation and attenuates adverse cardiac remodeling after MI in mice. The overexpression of the natural antagonist of the IL-1β, IL1RA, or the administration of a recombinant form of human IL1RA, anakinra, decreases postinfarction cardiac remodeling. On the other hand, antiangiogenic interleukins may exert beneficial effects on postischemic cardiac remodeling. IL-10 inhibits postischemic revascularization,27,28 but exerts a protective effect in postischemic cardiac remodeling.30 These results indicate that therapeutic strategies aimed at regulating inflammation should target a specific type (or subtype) of inflammatory cells, and evaluate not only short-term, but also long-term effects of inflammatory mediators on both vascular and tissue regeneration. Interestingly, nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly6Chigh monocyte subset traffic, reduce infarct inflammation and improve ejection fraction in apoE−/− mice after MI.31





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Keywords: atherosclerosis; B lymphocyte; inflammation; interleukin; ischemia; T lymphocyte