Introduction
Atherosclerosis is a chronic inflammatory process triggered by accumulation of cholesterol-containing low-density lipoprotein (LDL) particles in the arterial wall. Major etiologic factors include hyperlipidemia, hypertension, diabetes, and cigarette smoking, all of which are thought to initiate and promote vascular inflammation. The notion of atherosclerosis as an inflammatory disease has emerged based on observations of immune activation and inflammatory signaling in human atherosclerotic lesions, inflammatory biomarkers as independent risk factors for cardiovascular events, and an LDL-induced immune activation.
The use of animal models of atherosclerosis, such as hyperlipidemic rabbits and mice lacking either apolipoprotein E (ApoE -/- ) or the LDL receptor (LDLr -/- ), has provided major mechanistic insight into the basic mechanisms of atherosclerosis.
Initiation of Atherosclerosis
Atherosclerosis is initiated by the infiltration of apolipoprotein B (apoB)-containing LDL in the arterial wall ( Fig. 4.1 ). Atherosclerotic lesions preferentially occur in arterial bifurcations and when the caliber of the arterial tree changes. The switch from a laminar longitudinal to a turbulent flow at those sites will lead to a local recirculation and consequently increased concentrations of plasma LDL adjacent to the luminal surface. As a result, an increased radial LDL transport will occur into the arterial wall, where LDL can be retained by proteoglycans. Endothelial cells are sensitive to shear stress and the frictional force generated by blood flow. Whereas the normal laminar shear stress may be atheroprotective, a disturbed flow activates proinflammatory transcriptional programs in endothelial cells, which participate in the initiation of the inflammatory reaction at sites prone to develop atherosclerotic lesions. In addition, endothelial dysfunction hampers the barrier function of this cell layer, leading to increased influx of cholesterol-containing lipoproteins into the arterial intima.
Modification of the retained LDL by, for example, oxidation, may serve as an initiating stimulus for inflammatory reactions, by being recognized as a so-called danger-associated-molecular-pattern (DAMP). Specific pattern recognition, such as toll-like receptor (TLR) activation by oxidized LDL, subsequently stimulates endothelial cells to express adhesion molecules. Oxidatively modified LDL particles induce endothelial cell surface expression of leukocyte adhesion molecules such as E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1, which bind to their ligands sialyl-Lewis X , integrins CD11/18, and VLA-4, expressed on leukocytes. The combinatorial expression of endothelial adhesion molecules and leukocyte integrins and selectins provides a sophisticated regulation of the inflammatory process and determines the type and place for recruitment of a certain type of myeloid or lymphoid cell during atherosclerosis development as depicted in Fig. 4.1 .
Atherosclerotic Inflammation and Immune Activation
The leukocytes recruited to the developing atherosclerotic lesion produce a number of inflammatory mediators ( Fig. 4.2 ) that will amplify the inflammatory reaction through a continued activation of both leukocytes and endothelial cells and by recruiting further immune cells to the forming lesion. These mediators are further discussed hereafter.
Monocytes represent the most numerous white blood cells recruited into atherosclerotic plaques. Once resident in the arterial wall, they differentiate into tissue macrophages under the influence of monocyte-colony stimulating factor (M-CSF) present in forming lesions. Activated macrophages in the atherosclerotic lesion further enrich the proinflammatory milieu, by means of inflammatory proteins and lipid mediators, such as cytokines and leukotrienes. This subtype, which is referred to as classically activated or M1 macrophages , will hence sustain inflammatory responses and result in tissue damage. In contrast, the alternatively activated, or M2, macrophages secrete antiinflammatory mediators such as lipoxin (LX) A 4 , interleukin (IL)-10, and transforming growth factor (TGF)-β and may promote the resolution of inflammation by means of clearance of apoptotic cells (efferocytosis) and dampening of immune responses, hence promoting tissue repair and healing. Both M1 and M2 macrophages are present at different stages of human atherosclerotic plaque development, and data suggest that the atherosclerotic lesion macrophages constitute a unique subset. This may necessitate further subclass characterization depending on their specific functions and signaling pathways, although it should be kept in mind that the macrophage is a highly plastic cell that can modulate its phenotype depending on its local environment.
The number of mast cells is low in the normal vessel. However, mast cell numbers increase with lipid accumulation in the vascular wall in early atherosclerosis, implying that mast cell progenitors are recruited from the arterial lumen.
At this stage, with the presence of both retained naïve and modified LDL, together with activated leukocytes, the atherosclerotic lesion is emerging. Oxidized and otherwise modified forms of LDL particles can bind to scavenger receptors, such as SRA-1, CD36, and LOX-1, all of which are expressed on resident macrophages. The resulting uptake of lipoprotein particles will induce the conversion of macrophages into foam cells, a pathogenic process that results in the microscopic appearance of lipid-laden macrophages, which is a characteristic of the atherosclerotic lesion.
The internalization of oxidized LDL (oxLDL) by macrophages and dendritic cells will lead not only to foam cell formation, but also antigen presentation. The processing of modified lipoproteins and other antigens followed by a subsequent presentation to T cells will hence activate the adaptive immune system within the atherosclerotic lesion. Although oxidation of LDL has been thought to be the source of neoantigens, this hypothesis has been challenged by results showing that T cells in atherosclerotic mice recognize peptide motifs of native LDL particles and its ApoB100 moiety. This suggests that cellular immunity toward LDL as an autoantigen might drive atherosclerosis.
Effector CD4+ T cells are recruited to the atherosclerotic lesion by leukocyte adhesion molecules and chemotactic factors produced as a consequence of innate immune activation. In addition to Th1 cells, effector T cells of the Treg subtype are present in atherosclerotic lesions and act by inhibiting immune responses and inflammation; hence they are considered as atheroprotective. The Th17 cell subtype, finally, promotes fibrosis through action of its signature cytokine, IL-17. Therefore, Th17 activity enhances formation of the lesion’s fibrous cap and, hence, presumably, plaque stability.
Several factors in the atherosclerotic lesions induce macrophage apoptosis. Under normal conditions, apoptotic cells are cleared by a specific phagocytosis process, termed efferocytosis, from the Greek word “to bury.” Efferocytosis is an immune response essential for normal steady state of a tissue and a critical phenomenon in the resolution of inflammation. Defective clearance of lipid-laden apoptotic macrophages in the atherosclerotic lesion will create a lipid necrotic core, as depicted in Fig. 4.1 .
In addition to the previously mentioned inflammatory circuits, which take place in the intima, complex adaptive immune responses also develop in the adventitia and the periadventitial connective tissues. Antigens reach the adventitia via the vasa vasorum and also through convection of macromolecules from the arterial lumen. Inflammatory cells observed in the adventitia of atherosclerotic lesions include dendritic cells, macrophages, mast cells, and lymphocytes. T and B cell activation is present in the adventitia of atherosclerotic vessels, and in advanced stages of atherosclerosis, large lymphoid structures may develop, referred to as adventitial tertiary lymphoid organ s (see Fig. 4.1 ). The latter contain germinal centers with B cells going through differentiation to centrocytes and plasma cells. Surrounding them, dendritic cells, T cells, and macrophages form organized structures of interacting cells. These adventitial tertiary lymphoid organs are sites of antibody production, including antibodies to plasma lipoproteins. Interestingly, deposits of ceroid-containing oxidized lipids are also found here, suggesting that they may serve as antigenic stimuli for antibody production.
Inflammatory and Antiinflammatory Proteins
Cytokines
In the 1980s, IL-1 was identified as a cytokine of the vasculature, regulating hemostatic properties and leukocyte adhesion. The discovery that macrophages initiate IL-1β production as a response to cholesterol accumulation by means of a multiprotein oligomer called the inflammasome and the development of IL-1β-neutralizing antibodies for clinical use have renewed the interest in this cytokine in the context of atherosclerosis, as discussed later. Other cytokines that have been studied for their proatherogenic role include tumor necrosis factor (TNF), interferon (IFN)-γ, and IL-6, as well as others listed in Fig. 4.2 . Retrospective analysis of studies of rheumatoid arthritis patients receiving TNF blockade has strengthened the importance of cytokine signaling in atherosclerosis, because these patients exhibit a decreased cardiovascular risk compared with those given alternative treatment. These observations reinforce the notion of TNF being an important proinflammatory signaling factor in atherosclerosis and have suggested that TNF blockade may be useful for cardiovascular prevention.
Several lines of experimental evidence implicate IFN-γ, the signature cytokine of Th1 cells, as a powerful proatherosclerotic cytokine. IFN-γ increases lesion development, modulates lipoprotein metabolism, and inhibits fibrous cap formation. Its presence in culprit lesions of human atherosclerosis supports the notion that Th1/IFN-γ activity may be deleterious in atherosclerosis.
IL-6 is produced in large amounts by IL-1-stimulated cells, including vascular and blood-derived ones. As large amounts of IL-6 are produced by IL-1-stimulated cells, this cytokine acts as an amplifier of vascular inflammation, and circulating IL-6 levels have been reported to predict clinical events. When IL-6 reaches the liver, it induces an acute-phase response that involves increased production of C-reactive protein and fibrinogen and subsequent higher circulating levels of these acute-phase reactants. Therefore, C-reactive protein measurement has become an attractive way of estimating atherosclerosis-associated inflammation.
In contrast to these proinflammatory cytokines, TGF-β and IL-10, produced by M2 macrophages and Tregs, activate suppressive pathways and have antiatherosclerotic effects (see Fig. 4.2 ). Finally, IL-17 produced by Th17 cells may both increase atherosclerosis formation and promote collagen synthesis, which stabilizes the atherosclerotic lesion.
Chemokines
Chemokines are a specific family of chemotactic proteins, classified in subgroups based on the position of the N-terminal cysteine residues (CC, CXC, CX3C, XC). Several studies have supported a key role of chemokines in atherosclerosis by means of mediating immune cell recruitment and regulating the activation of different immune cell types and subsets. Endothelium-derived CXCL1 and monocyte chemoattractant protein 1 (MCP-1, also referred to as CCL2) are involved in early atherosclerosis by means of specific chemokine receptors. In addition, the chemokine-like protein migration inhibitory factor (MIF) also binds to chemokine receptors (CXCR2 and CXCR4) to mediate monocyte and T lymphocyte recruitment to atherosclerotic lesions. Inhibiting MCP-1 binding to CCR2 reduces inflammatory biomarkers in subjects with cardiovascular risk factors, supporting the importance of chemokine signaling as a regulator of inflammation in atherosclerosis (see Fig. 4.2 ).
In contrast to the previously mentioned proinflammatory chemokine-induced effects, other chemokines, such as CCL19/CCL21, CXCL5, and CXCL12, mediate macrophage regression from atherosclerotic lesions, block foam cell formation, improve endothelial repair, and increase plaque stability under certain conditions, illustrating that changes in chemokine profiles may drive the atherosclerotic lesion to either progression or regression (see Fig. 4.2 ).
Lipid Mediators of Inflammation and Resolution
In addition to the aforementioned proteins, bioactive lipids (see Fig. 4.2 ) provide important signaling in atherosclerosis. Their generation may derive from either extracellular metabolism of phospholipids from circulating lipoproteins or intracellular enzymatic pathways using membrane phospholipids as substrate.
Phospholipases
The hydrolysis of phospholipids into fatty acids by the phospholipase A 2 (PLA 2 ) family of enzymes releases arachidonic acid and lysophospholipids. The secreted sPLA 2 has been detected in human atherosclerotic lesions and participates in LDL modification by hydrolysis of phosphatidylcholine, hence rendering the LDL molecule more atherogenic ( Fig. 4.3 ). Another PLA 2 isoenzyme, LpPLA2, which hydrolyzes oxidized phospholipids in LDL particles to proinflammatory lysophosphatidylcholine and oxidized nonesterified fatty acids (oxNEFAs), has also been identified as a risk marker for atherosclerosis (see Fig. 4.3 ). However, although apparently reducing atherosclerosis in animal models of atherosclerosis and surrogate markers in early clinical trials, large randomized clinical trials (RCTs) have not demonstrated any beneficial effects for PLA 2 inhibitors in terms of cardiovascular prevention.
The Cyclooxygenase Pathway
The two cyclooxygenase (COX) enzymes, COX-1 and COX-2, catalyze the formation of prostaglandins (PGs) and thromboxane (TX). The COX isoenzymes are the targets for nonsteroidal antiinflammatory drugs (NSAIDs). The use of low-dose aspirin in secondary prevention relies on its irreversible inhibition of COX-1 in platelets, which lack the ability to resynthesize COX enzymes, leading to a selective inhibition of platelet proaggregatory TXA 2 formation. In contrast to the constitutively expressed COX-1, the COX-2 isoform is induced by proinflammatory stimuli at sites of inflammation, such as atherosclerotic lesions. The use of NSAIDs being either selective or preferential for the COX-2 isoform (COX-2 inhibitors or coxibs) has however been associated with an increased cardiovascular risk in several RCTs and observational studies and has led to withdrawal and precautions in their prescription to subjects with an increased cardiovascular risk (see Bäck et al. and references therein). Despite potential antiinflammatory effects, the detrimental outcome of COX-2 inhibition in atherosclerosis may be due to a disturbed balance between TXA 2 and prostacyclin, which exert opposing effects in terms of platelet aggregation, pro- and antiatherogenic signaling, and alterations of vascular reactivity. However, other prostaglandins also affect several responses in the vascular wall and inflammatory cells with potential importance for atherosclerosis, and the balance of the COX pathway, both locally in atherosclerotic lesions and systemically, may be more complex (see Fig. 4.3 ).
The Lipoxygenase/Leukotriene Pathways
Arachidonic acid also serves as a substrate for the 5-lipoxygenase (5-LO) enzyme and leukotriene (LT) biosynthesis (see Fig. 4.3 ). Arachidonic acid metabolism by the 5-LO enzyme together with the 5-LO activating protein (FLAP) leads to the formation of the unstable LTA 4 , which subsequently is either hydrolyzed into the dihydroxy LTB 4 or conjugated with glutathione to yield the cysteinyl-LTs (LTC 4 , LTD 4, and LTE 4 ). These LTs act on specific receptors, BLT and CysLT receptors, respectively, to transduce several proinflammatory effects with implications for atherosclerosis development, such as leukocyte recruitment and activation, smooth muscle cell (SMC) proliferation, and endothelial dysfunction. Local LT biosynthesis and expression of LT forming enzymes are detected in human atherosclerotic lesions, and biomarker studies have associated LTs with acute coronary syndromes and subclinical atherosclerosis. Genetic or pharmacologic targeting of 5-LO and FLAP has, however, generated contradictory results in terms of atherosclerosis development in hyperlipidemic mouse models. Nevertheless, antileukotrienes that are in clinical use for the treatment of asthma and allergic rhinitis have been associated with a reduced risk of recurrent cardiovascular events in retrospective analysis.
Specialized Proresolving Mediators
In addition to the formation of proinflammatory leukotrienes, lipoxygenases also participate in the formation of antiinflammatory lipid mediators, which participate in the resolution of inflammation. For example, arachidonic acid metabolism by means of dual lipoxygenation leads to the formation of lipoxin A 4 (LXA 4 ) (see Fig. 4.3 ), whereas the metabolite resulting from the consecutive action of LO and COX-2 acetylated by aspirin is an LXA 4 analogue termed aspirin-triggered lipoxin (ATL). These lipoxins are produced locally in coronary atherosclerotic lesions, and their levels increase after aspirin treatment.
In addition to arachidonic acid, omega-3 fatty acids can serve as the substrate for lipoxygenase metabolism, yielding a number of bioactive lipids, such as resolvins, maresins, and protectins, which also promote the resolution of inflammation. Fish oil supplementation to ApoE -/- mice leads to increased incorporation of the omega-3 fatty acids docosahexeanoic acid (DHA) and eicosapentaenoic acid (EPA) in cell membranes of different organs, but the effects on atherosclerosis have been somewhat varying between different studies, time points, and models.
Lipoxins and resolvins stimulate efferocytosis, which is an important mechanism of the resolution of inflammation and may serve to decrease the necrotic core formation in atherosclerosis (see previous section and Fig. 4.1 ). LXA 4 , ATL, and RvD1 exert their proresolving actions by means of the receptor FPR2/ALX (formyl peptide receptor 2 and A type lipoxin receptor), which is also activated by a number of proinflammatory agonists in the atherosclerotic lesion. Studies in human carotid atherosclerotic plaques and animal models have implicated this receptor in atherosclerosis progression and plaque stability. In addition to macrophages, the FPR2/ALX receptor is also expressed on vascular SMCs, and ATL inhibits vascular SMC migration and proliferation, suggesting additional benefits of aspirin in the treatment of coronary atherosclerosis.
A link between omega-3 fatty acids and decreased cardiovascular inflammation was first observed in Greenland Inuits. The higher levels of DHA and EPA in the plasma and platelets of Inuits compared with other Scandinavians were inversely related to population rates of acute myocardial infarction. Epidemiologic and clinical trial evidence subsequently accumulated in further support of antiinflammatory effects for omega-3 fatty acids. However, the effects of omega-3 supplementation in secondary prevention of coronary artery disease have not been consistently replicated, and further studies are ongoing. The structural elucidation of the active proresolution omega-3-derived mediators as resolvins, maresins, and protectins (see Figs. 4.2 and 4.3 ) suggests specific stimulations of these pathways as putative therapeutic options in atherosclerosis.
Intracellular Inflammatory Signaling Pathways
p38 Mitogen-Activated Protein Kinase
Among the intracellular pathways that transduce the cellular responses to the extracellular proinflammatory stimuli discussed previously, phosphorylation cascades play a key role in regulating cellular activity ( Fig. 4.4 ). The p38 serine kinase is one of the mitogen-activated protein kinase (MAPK) pathways transducing and amplifying intracellular inflammatory responses, such as cytokine production (IL-1β, TNF, and IL-6) and enzyme activity (COX-2-derived PGE 2 formation). Furthermore, p38 phosphorylation may be inhibited by specialized proresolving mediators as part of their role in the resolution of inflammation. Recent clinical evaluations have suggested a potential antiinflammatory and therefore beneficial effect of p38 inhibitors in the treatment of atherosclerosis.
