**2. Inflammatory mediators in atherosclerosis**

The evidence for atherosclerosis as an inflammatory disease is solid. The importance of immune activation in atherosclerosis is demonstrated in several animal models, were removal of central inflammatory mediators or cell types have been shown to extensively reduce plaque development [9–11]. Further, both communicable and noncommunicable inflammatory conditions increase the risk of cardiovascular disease (CVD), and CRP is an independent risk factor of cardiovascular events, both in healthy individuals and in patients with established disease [12–14]. Moreover, immune cells are present within all atherosclerotic plaques, from early fatty streaks to complex atheromas. Lesional inflammation increases during the course of plaque development and is most prominent in vulnerable plaques with large necrotic cores. Immune cells, but also smooth muscle cells, platelets and endothelial cells are drivers of plaque inflammation. Further, there are numerous different inflammatory triggers contributing to the great complexity of atherosclerotic plaque inflammation.

#### **2.1. Immune cells in atherosclerotic inflammation**

#### *2.1.1. Macrophages: linking lipid metabolism and inflammation in atherogenesis*

Macrophages are involved in all stages of plaque development, and are the most important immune cell in atherogenesis. Monocytes originate from a common stem cell in the bone marrow and migrate to various tissues where they develop into tissue-specific macrophages. Although circulating monocytes are a heterogeneous population, it is not known if distinct monocyte subtypes develop into specific macrophage subtypes in humans. Monocytes from the circulation are recruited to the intimal layer of an artery by chemokines (e.g., CCL2) and neuronal guidance molecules (e.g., ephrin-B2). Inside the plaque, the monocytes differentiate to macrophages and engulf modified lipids through scavenger receptors such as SR-A1 and CD36. These lipid-filled macrophages, called foam cells, have altered phenotype and immune function. The efferocytotic capacity (ability to clear apoptotic cells) is one of the functions affected, and as extensive cholesterol accumulation is also lethal to the cells, a necrotic core consisting of cell debris and lipids forms inside the lesion. Plaques with large necrotic cores are associated with an unstable plaque phenotype and are prone to rupture. Monocyte infiltration and foam cell formation are key elements in plaque development and provide the main link between lipid metabolism and chronic inflammation. There is, however, not only the quantity but also the phenotype of the macrophages that is important to the fate of the plaque [15]. The terms M1 and M2 describe the "classical" activated macrophage induced by T helper cell (Th) 1 cytokine interferon (IFN) γ and the "alternatively" activated macrophage induced by Th2 cytokines IL-13 and IL-4, respectively. In short, the M1 macrophages produce pro-inflammatory cytokines and chemokines, cause tissue injury and promote atherosclerotic plaque development. M2 macrophages are often divided into "wound healing" and "regulatory" macrophages, the latter induced by immune complexes and IL-10, and produce anti-inflammatory cytokines and increase plaque stability [16]. Their pro- and antiatherogenic role is supported by studies showing that plaques enriched in M2 macrophages are associated with a stable or regressive phenotype and vice versa. Growth factors, lipids and cytokines produced by vascular cells and immune cells in the plaque affect the macrophage polarization state. Due to the complexity of inflammatory stimuli present in the plaque, the terms "M1" and "M2" and "classical" and "alternative" are overly simplified, and it is more likely that there exists a range of overlapping phenotypes in the atherosclerotic lesions [15, 17–19].

#### *2.1.2. Dendritic cells are professional antigen presenting cells in the plaque*

Another cell of the innate immune system, with great importance for atherosclerotic plaque inflammation, is the dendritic cell (DC). Increased number of DCs is present in atherosclerotic plaques of both humans and mice, and also, as described later, in tertiary lymphoid organs in the adventitia. However, the circulating number of DCs has, by the majority of studies performed, been reported to be reduced in atherosclerosis, which could reflect hampered production from the bone marrow as well as increased recruitment to the plaque [20–22]. As macrophages, the DCs engulf lipids and become foam cells, thereby contributing to plaque development. On the contrary, it has also been suggested that DCs can control cholesterol homeostasis and counteract hypercholesterolemia. It is, however, their role as antigen presenting cells (APCs) that is most described in plaque inflammation [22]. Antigen presentation to T cells occurs both inside the plaque and in the lymphatic tissue, and it is shown that DCs can leave the atherosclerotic lesion upon signals from the chemokines CCL19 and CCL21 [23]. The different subgroups of DCs activate pro- and anti-inflammatory functions in T cells. Difficulties in finding DC-specific markers, as well as the broad spectrum of different DC cell subtypes, have complicated the study of DCs in atherogenesis. There is, however, without doubt that DCs are important players in atherosclerotic disease [22, 24].

#### *2.1.3. Other innate immune cells in atherogenesis*

Neutrophils, mast cells and innate lymphoid cells, such as natural killer (NK) cells, are also important contributors to inflammation in atherogenesis, and their role is increasingly appreciated. The description of these cell types is beyond the scope of this chapter, but has been reviewed elsewhere [25–28].

#### *2.1.4. T-cell diversity in atherogenesis*

evolved extensively and paved the way for in-depth understanding of how the immune system works. Identification of adhesion markers on endothelial cells and thus the ability of leukocytes to migrate into atheromas gave plausibility of inflammation as a contributor in atherogenesis [2, 3]. Furthermore, findings showing that monocytes [4], and later on that vascular cells [5, 6], secrete inflammatory mediators were important evidence supporting this. These discoveries were followed by clinical proof in the 1990s. Immune activation in atherosclerotic plaques was identified [7], and myocardial infarction was recognized as a potent trigger of CRP release [8]. Since then, an extensive number of animal as well as clinical studies have established inflammation as a major driver of atherosclerotic disease. Regardless of this acceptance, our under-

The evidence for atherosclerosis as an inflammatory disease is solid. The importance of immune activation in atherosclerosis is demonstrated in several animal models, were removal of central inflammatory mediators or cell types have been shown to extensively reduce plaque development [9–11]. Further, both communicable and noncommunicable inflammatory conditions increase the risk of cardiovascular disease (CVD), and CRP is an independent risk factor of cardiovascular events, both in healthy individuals and in patients with established disease [12–14]. Moreover, immune cells are present within all atherosclerotic plaques, from early fatty streaks to complex atheromas. Lesional inflammation increases during the course of plaque development and is most prominent in vulnerable plaques with large necrotic cores. Immune cells, but also smooth muscle cells, platelets and endothelial cells are drivers of plaque inflammation. Further, there are numerous different inflammatory triggers contributing to the great

Macrophages are involved in all stages of plaque development, and are the most important immune cell in atherogenesis. Monocytes originate from a common stem cell in the bone marrow and migrate to various tissues where they develop into tissue-specific macrophages. Although circulating monocytes are a heterogeneous population, it is not known if distinct monocyte subtypes develop into specific macrophage subtypes in humans. Monocytes from the circulation are recruited to the intimal layer of an artery by chemokines (e.g., CCL2) and neuronal guidance molecules (e.g., ephrin-B2). Inside the plaque, the monocytes differentiate to macrophages and engulf modified lipids through scavenger receptors such as SR-A1 and CD36. These lipid-filled macrophages, called foam cells, have altered phenotype and immune function. The efferocytotic capacity (ability to clear apoptotic cells) is one of the functions affected, and as extensive cholesterol accumulation is also lethal to the cells, a necrotic core consisting of cell debris and lipids forms inside the lesion. Plaques with large necrotic cores are associated with an unstable plaque phenotype and are prone to rupture. Monocyte infiltration and foam cell formation are key elements in plaque development and provide the main link between lipid metabolism and

standing of atherogenic inflammation is far from complete.

32 Atherosclerosis - Yesterday, Today and Tomorrow

**2. Inflammatory mediators in atherosclerosis**

complexity of atherosclerotic plaque inflammation.

**2.1. Immune cells in atherosclerotic inflammation**

*2.1.1. Macrophages: linking lipid metabolism and inflammation in atherogenesis*

CD4+ Th cells are the most abundant of the adaptive immune cells in the plaque and are therefore the most studied. In the plaque, they are activated by epitopes of native as well as oxidative LDL presented by antigen-presenting cells (i.e., DCs). Activated T cells can affect atherosclerosis in two ways: through effector functions in the arterial wall and by activating B cells in lymphoid organs to produce circulating antibodies [29]. For CD4+ Th cells, several subsets have been identified. Most is known about the role of Th1 and Th2 in atherosclerosis; however, in recent years, it has become evident that Th17 and Tregs also are important players in atherogenesis. Polarization of Th cells is determined by the cytokine environment, and the proinflammatory Th1 cells are the most abundant T cell in the plaques. Th1 is characterized by secretion of IFN-γ, and Th2 typically secretes IL-4, IL-5 and IL-13. Th17 secretes IL-17 and IL-22, and Tregs secrete IL-10 and transforming growth factor (TGF)-β. In short, the Th1 cells are proatherogenic, while Tregs are atheroprotective. The impact of Th2, Th17 and natural killer T cells (NKTs) on atherosclerosis has shown more conflicting results, but are all present in the plaque. CD8+ cytotoxic T cells are also present in atherosclerotic lesions, although less frequent than CD4+ effector cells. Their activation and importance in atherosclerosis is not completely understood, but they can exert proatherogenic effects through IFN-γ production and macrophage activation or through their cytotoxic activity. Recently, the CD8<sup>+</sup> regulatory T cell was described, with possible atheroprotective effects, through modulatory effects on T cell–B cell interaction [30, 31].

adventitia by production of the lymphorganogenic chemokines CXCL13 and CCL21 [42, 43]. The TLOs have a different composition of immune cells than the macrophage-rich plaques and is mostly composed of dendritic cells, T cells and a high number and diversity of B cells [43, 44]. This supports the role of TLOs as sites for T-cell training [45] and activation of local humoral immune responses [37]. A recent paper suggests that TLOs participate in atheroprotection [45], however, as the plaque itself, the TLOs can harbor both pro- and anti-inflammatory mediators,

Inflammatory Mechanisms in Atherosclerosis http://dx.doi.org/10.5772/intechopen.72222 35

Plaque development does not occur randomly, but typically at curvatures and branching points in the arteries. At these sights, the shear stress activates the endothelial cells lining the arterial wall, leading to structural, molecular and functional alterations in the cells. Atheroprone flow activates the Nf-Kβ pathway and TLR2 expression in endothelial cells as well as a spectrum of other conduits leading to increased endothelial proliferation and inflammation. The activated endothelial cells adhere leukocytes and stimulate neighboring cells, e.g. vascular smooth muscle cells (VSMCs) [47–49]. Upon atherogenic stimuli, that is, from the activated endothelium, VSMCs undergo so-called phenotype switching. They progress from quiescent contractile to proliferative and migratory cells. These cells possess atheroprotective functions, as they produce extracellular matrix and proteoglycans, which protects the plaque from rupture. They do, however, also accumulate lipids and become macrophage-like foam cells, contributing to plaque development [50, 51]. Further, they express adhesion molecules such as VCAM-1 and ICAM-1 and thereby contribute to retention of monocytes and macrophages in the lesions [52, 53]. Thus, VSMCs can have both protective and destructive effects, depending on the stage of plaque development, and the stimuli present. Inflammatory monocytes can further stimulate VSMCs to secrete proatherogenic matrix metalloproteinases (MMPs), which increase the risk of plaque rupture through thinning of the fibrous cap [54]. Further, VSMCs produce a variety of cytokines, activating immune cells, endothelial cells and other VSMCs in the lesion [51]. The inflammatory, atheroprone contribution of VSMCs is however probably under-communicated, as lack of cell-specific markers complicates their identification. Macrophages can express "classical" smooth muscle cell markers (i.e., α-actin and SM22α), and vice versa (i.e., CD68 and Mac2), and this is determined by the presence of lipids and inflammatory stimuli in the plaque [55, 56]. The local inflammatory micro milieu will therefore decide the inflammatory contribution of smooth muscle cells to atherogenesis by regulating the transition of VSMC into inflammatory cells. Thus, there is a need for better markers to more correctly determine

Also nonimmune cells in the circulation can contribute to the inflammatory milieu during atherogenesis. In addition to their most known roles as blood clotting cells, platelets also possess a great inflammatory potential. In a bidirectional manner, platelets interact with both leukocytes and endothelial cells to communicate inflammation. They express a variety of inflammatory mediators and receptors and contribute to atherosclerotic inflammation throughout disease development, from development of fatty streaks to thrombus formation.

For an extensive review of the role of platelets in atherogenesis, see [57].

and thus, the net effect of adventitial inflammation is still elusive [46].

the role of VSMCs in atherosclerotic inflammation.

**2.3. Inflammatory mechanisms of nonimmune cells in atherosclerosis**

#### *2.1.5. B cells and atherogenic antibodies*

B cells are divided into two subtypes: B1 and B2 cells, and both of these are involved in atherogenesis. B1 cells are involved in innate humoral immune response, divide upon self-renewal in the periphery and produce antibodies with low specificity. In contrast, B2 cells are conventional B cells, which differentiate to plasma cells upon antigen presentation by T cells and DCs in lymph nodes, producing antibodies with high affinity and thereby contribute in adaptive immunity [30, 32]. Several animal models with B cell depletion resulting in aggravation of atherosclerosis have suggested a protective role for B cells in atherogenesis [33, 34]. Specific depletion of B2 cells has, however, been shown to reduce the development of atherosclerosis, suggesting subset specificity with regard to B cell atherogenity [35, 36]. In contrast to B2 cells which are mainly proatherogenic, producing IgG antibodies and activating T cells, B1 cells produce IgM antibodies, which can bind and thereby block the uptake of oxLDL by macrophages, exerting atheroprotective effects [37]. Most of these studies are performed in animal models, and thus, the importance for B cells in human atherosclerosis is unclear. In contrast to macrophages and T cells, B cells are only found in some atherosclerotic plaques, and a more abundant in so-called tertiary lymph organs, in the adventitial layer of the artery.

#### **2.2. Tertiary lymphoid organs: extended plaque inflammation**

During chronic inflammatory conditions, lymph-node–like structures, termed tertiary lymphoid organs (TLOs), can develop. Immune cells in the adventitial layer of atherosclerotic arteries were discovered decades ago [38], but the importance of these TLOs for atherosclerotic plaque inflammation is still unknown. Advanced plaques are, however, associated with increased adventitial inflammation in both humans [39, 40] and Apoe−/− mice [41], suggesting that such extended plaque inflammation is important in the disease process. They likely evolve as a response to arterial wall inflammation in early lesion development. Medial SMCs are suggested as drivers of TLO development and are upon inflammatory stimuli shown to attract immune cells into adventitia by production of the lymphorganogenic chemokines CXCL13 and CCL21 [42, 43]. The TLOs have a different composition of immune cells than the macrophage-rich plaques and is mostly composed of dendritic cells, T cells and a high number and diversity of B cells [43, 44]. This supports the role of TLOs as sites for T-cell training [45] and activation of local humoral immune responses [37]. A recent paper suggests that TLOs participate in atheroprotection [45], however, as the plaque itself, the TLOs can harbor both pro- and anti-inflammatory mediators, and thus, the net effect of adventitial inflammation is still elusive [46].

#### **2.3. Inflammatory mechanisms of nonimmune cells in atherosclerosis**

LDL presented by antigen-presenting cells (i.e., DCs). Activated T cells can affect atherosclerosis in two ways: through effector functions in the arterial wall and by activating B cells in lymphoid organs to produce circulating antibodies [29]. For CD4+ Th cells, several subsets have been identified. Most is known about the role of Th1 and Th2 in atherosclerosis; however, in recent years, it has become evident that Th17 and Tregs also are important players in atherogenesis. Polarization of Th cells is determined by the cytokine environment, and the proinflammatory Th1 cells are the most abundant T cell in the plaques. Th1 is characterized by secretion of IFN-γ, and Th2 typically secretes IL-4, IL-5 and IL-13. Th17 secretes IL-17 and IL-22, and Tregs secrete IL-10 and transforming growth factor (TGF)-β. In short, the Th1 cells are proatherogenic, while Tregs are atheroprotective. The impact of Th2, Th17 and natural killer T cells (NKTs) on atherosclerosis has shown more conflicting results, but are all present in the plaque. CD8+ cytotoxic T cells are also present in atherosclerotic lesions, although less frequent than CD4+ effector cells. Their activation and importance in atherosclerosis is not completely understood, but they can exert proatherogenic effects through IFN-γ production and macrophage activation or through

protective effects, through modulatory effects on T cell–B cell interaction [30, 31].

abundant in so-called tertiary lymph organs, in the adventitial layer of the artery.

During chronic inflammatory conditions, lymph-node–like structures, termed tertiary lymphoid organs (TLOs), can develop. Immune cells in the adventitial layer of atherosclerotic arteries were discovered decades ago [38], but the importance of these TLOs for atherosclerotic plaque inflammation is still unknown. Advanced plaques are, however, associated with increased adventitial inflammation in both humans [39, 40] and Apoe−/− mice [41], suggesting that such extended plaque inflammation is important in the disease process. They likely evolve as a response to arterial wall inflammation in early lesion development. Medial SMCs are suggested as drivers of TLO development and are upon inflammatory stimuli shown to attract immune cells into

**2.2. Tertiary lymphoid organs: extended plaque inflammation**

B cells are divided into two subtypes: B1 and B2 cells, and both of these are involved in atherogenesis. B1 cells are involved in innate humoral immune response, divide upon self-renewal in the periphery and produce antibodies with low specificity. In contrast, B2 cells are conventional B cells, which differentiate to plasma cells upon antigen presentation by T cells and DCs in lymph nodes, producing antibodies with high affinity and thereby contribute in adaptive immunity [30, 32]. Several animal models with B cell depletion resulting in aggravation of atherosclerosis have suggested a protective role for B cells in atherogenesis [33, 34]. Specific depletion of B2 cells has, however, been shown to reduce the development of atherosclerosis, suggesting subset specificity with regard to B cell atherogenity [35, 36]. In contrast to B2 cells which are mainly proatherogenic, producing IgG antibodies and activating T cells, B1 cells produce IgM antibodies, which can bind and thereby block the uptake of oxLDL by macrophages, exerting atheroprotective effects [37]. Most of these studies are performed in animal models, and thus, the importance for B cells in human atherosclerosis is unclear. In contrast to macrophages and T cells, B cells are only found in some atherosclerotic plaques, and a more

regulatory T cell was described, with possible athero-

their cytotoxic activity. Recently, the CD8<sup>+</sup>

*2.1.5. B cells and atherogenic antibodies*

34 Atherosclerosis - Yesterday, Today and Tomorrow

Plaque development does not occur randomly, but typically at curvatures and branching points in the arteries. At these sights, the shear stress activates the endothelial cells lining the arterial wall, leading to structural, molecular and functional alterations in the cells. Atheroprone flow activates the Nf-Kβ pathway and TLR2 expression in endothelial cells as well as a spectrum of other conduits leading to increased endothelial proliferation and inflammation. The activated endothelial cells adhere leukocytes and stimulate neighboring cells, e.g. vascular smooth muscle cells (VSMCs) [47–49]. Upon atherogenic stimuli, that is, from the activated endothelium, VSMCs undergo so-called phenotype switching. They progress from quiescent contractile to proliferative and migratory cells. These cells possess atheroprotective functions, as they produce extracellular matrix and proteoglycans, which protects the plaque from rupture. They do, however, also accumulate lipids and become macrophage-like foam cells, contributing to plaque development [50, 51]. Further, they express adhesion molecules such as VCAM-1 and ICAM-1 and thereby contribute to retention of monocytes and macrophages in the lesions [52, 53]. Thus, VSMCs can have both protective and destructive effects, depending on the stage of plaque development, and the stimuli present. Inflammatory monocytes can further stimulate VSMCs to secrete proatherogenic matrix metalloproteinases (MMPs), which increase the risk of plaque rupture through thinning of the fibrous cap [54]. Further, VSMCs produce a variety of cytokines, activating immune cells, endothelial cells and other VSMCs in the lesion [51]. The inflammatory, atheroprone contribution of VSMCs is however probably under-communicated, as lack of cell-specific markers complicates their identification. Macrophages can express "classical" smooth muscle cell markers (i.e., α-actin and SM22α), and vice versa (i.e., CD68 and Mac2), and this is determined by the presence of lipids and inflammatory stimuli in the plaque [55, 56]. The local inflammatory micro milieu will therefore decide the inflammatory contribution of smooth muscle cells to atherogenesis by regulating the transition of VSMC into inflammatory cells. Thus, there is a need for better markers to more correctly determine the role of VSMCs in atherosclerotic inflammation.

Also nonimmune cells in the circulation can contribute to the inflammatory milieu during atherogenesis. In addition to their most known roles as blood clotting cells, platelets also possess a great inflammatory potential. In a bidirectional manner, platelets interact with both leukocytes and endothelial cells to communicate inflammation. They express a variety of inflammatory mediators and receptors and contribute to atherosclerotic inflammation throughout disease development, from development of fatty streaks to thrombus formation. For an extensive review of the role of platelets in atherogenesis, see [57].
