**5. Defective resolution in atherosclerosis**

**3. Inflammasome activation: a central inflammatory driver of** 

Inflammasomes are intracellular immune sensors, which are tightly controlled. They assemble upon stimuli from tissue damage, infection or metabolic disturbances, and their activation results in production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. There are several different inflammasomes, but the *NOD-like receptor containing a pyrin domain 3* (NLRP3) inflammasome is the most described and is an important constituent of innate immune apparatus. The NLRP3 inflammasome is a multimeric protein complex, which upon activation assembles and attracts caspase-1 molecules, which then is activated by self-cleavage. Active caspase-1 cleaves pro-IL⁻1β and pro-IL-18 to active cytokines ready for secretion [58, 59]. IL-1β is a prototypical proatherogenic cytokine, and NLRP3 is thus an important contributor to atherosclerotic inflammation. Cholesterol crystals, which deposit in atherosclerotic lesions, can activate the inflammasome both in vitro and in vivo [60, 61]. Further, the nonlipid danger signal ATP stimulates foam cell formation and cell migration through inflammasome activation [62]. Thus, the inflammasome promotes atherogenesis through inflammatory, as well as noninflammatory pathways, induced by lipid- as well as nonlipid stimuli. The NLRP3 inflammasome is present and activate in human atherosclerotic plaque [61]. Further, LDL receptor (LDLR)-deficient mice which received bone marrow from NLRP3-deficient mice show attenuated atherosclerosis, and silencing of NLRP3 in ApoE-deficient mice stabilizes atherosclerotic

plaques, pointing to an important role in atherosclerotic disease development [60, 63].

Inflammation is a part of the body's response to harm, either from microbes, such as virus and bacteria, from burns or toxins, or from injury. The main function is to eliminate the insult, remove damaged tissue and restore tissue homeostasis. In atherosclerosis, the signals of harm, termed *triggers*, are numerous. In contrast to infectious disease, the most typical triggers in atherogenesis are however sterile. These are termed damage-associated molecular patterns (DAMPs) and are host-derived danger signals released upon tissue damage, metabolic disturbances, or environmental stress. The risk factors of CVD include hyperlipidemia, smoking, hypertension and hyperglycemia, and all these factors cause DAMPs. There is, however, also evidence supporting a role for pathogens in atherosclerosis. These are termed pathogen-associated molecular patterns (PAMPs). Bacterial and viral microbes are found in atherosclerotic plaques and are associated with disease risk [64, 65]. In addition to pathogens, gut microbiota is a potential source of PAMPs, also linked to atherogenesis [66]. The causal relationship between the endogenous DAMPs and atherosclerosis is stronger than for PAMPs. Microbes do not seem to be required for atherogenesis, as germ-free mice are not protected against disease [67]. The DAMPs comprehend the necessary evil of atherogenesis, namely lipids. As mentioned, the interaction between lipids and immune activation is the hallmark of atherosclerotic disease. Nonmodified fatty acids can activate immune responses, and while saturated fats are shown to stimulate inflammation, polyunsaturated fats are repressors [68]. It is, however, the modified lipids that are the typical triggers during atherogenesis. In hyperlipidemia, LDL undergoes oxidation, forming oxidation-specific

**4. Danger signals in atherosclerosis**

**atherosclerosis**

36 Atherosclerosis - Yesterday, Today and Tomorrow

Inflammation is a beneficial process; however, it becomes detrimental if the response is too strong or too long. Cessation of inflammation was previously thought of as a passive process; however, it is now known that resolution of inflammation is a highly active process, involving a complex network of mediators. For inflammation to stay homeostatic, these mechanisms need to be intact. Atherosclerosis is characterized by a chronic nonresolving low-grade inflammation. Thus, a defective resolution of inflammation is an important contributor to atherosclerotic development and sustainability. Resolution is driven by endogenous specialized lipid-derived mediators (SPMs), which are synthesized from fatty acids, as well as proteins such as IL-10, M1 macrophages and the nucleotides inosine and adenosine. These stimulate tissue repair and regeneration and can therefore be distinguished from the classical anti-inflammatory signals, which are merely antagonists of pro-inflammatory signals. In a normal immune response, the production of SPMs is initiated by the production of the pro-inflammatory prostaglandins. Defective resolution in atherosclerosis can be summarized in three processes: (1) sustained inflammation, (2) increased infiltration/reduced egress of immune cells and (3) defective efferocytosis. In early atherosclerotic plaques, the efferocytotic capacity of macrophages (ability to clear apoptotic cells) is sufficient. Thus, inflammatory cells are cleared from the lesion, and this process elicits the release of anti-inflammatory mediators that counteract the plaque development. In advanced plaques, the efferocytotic capacity is however, as mentioned, compromised, leading to reduced clearance of dead cells, secondary necrosis and stimulation of the pro-inflammatory environment and growth of the necrotic core. SPMs are shown to counteract these processes and stabilize plaques [73, 77]. However, in advanced atherosclerotic lesions, the ratio of SPMs to pro-inflammatory mediators is decreased, and the administration of SPMs counteracts atherosclerotic disease development. These findings provide a mechanistic explanation for the defective resolution observed in atherosclerotic disease [78, 79]. To further map the production, regulation and function of pro-resolving mediators in atherosclerosis will be of great importance to increase our understanding of how the inflammation in atherosclerosis becomes nonresolving.

#### **6. Inflammation: a therapeutic target in atherosclerotic disease**

Despite the great success of modern treatment modalities, atherosclerosis is still the leading cause of mortality and morbidity worldwide. For example, high-dose statin treatment and other standard measures only prevent a fraction of recurrent events in survivors of MI. This residual burden of events presents a pressing unmet medical need, and novel perspective on atherogenesis is needed to treat those who are not met by the current treatment regimes. An interesting new therapeutics is the enzyme proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors. PCSK9 binds to the hepatic low-density lipoprotein (LDL) receptor, thereby inhibiting recycling of this receptor, resulting in attenuated removal of LDL cholesterol (LDL-C) from the circulation. The importance of PCSK9 for LDL-C homeostasis is illustrated in individuals with loss- and gain-of function mutations in this enzyme leading to hypo- or hypercholesterolemia, respectively, with dramatic effects on the incidence of atherosclerotic disease. Recent studies have shown that anti-PCSK9 therapies markedly reduce LDL-C levels, leading to lower incidence of adverse cardiovascular disease (CVD) outcomes in high-risk patients with hyperlipidemia [80]. In patients with LDL levels that remain above the treatment target, despite statin treatment (residual LDL risk); adding a PCSK9 inhibitor should be considered. Recent network meta-analysis demonstrates that PCSK9 inhibitors significantly reduce LDL cholesterol, on top of medium to high statin therapy [81]. Of direct relevance for inflammation, a very recent study, the CANTOS trial, suggests that those with residual inflammatory risk could benefit from interleukin-1β inhibition by Canakinumab. This anti-inflammatory treatment resulted in reduced cardiovascular risk, independent of lipid-lowering effects [82]. Another exciting approach to target inflammation in atherosclerosis is the pro-resolving mediators SPMs. In contrast to anti-inflammatory agents, these ligands will not compromise host defense, one of the most important challenges of immunosuppressive therapeutics. Many experimental studies have shown therapeutic potential for SPMs; however, more knowledge is needed to pinpoint how these mediators act, before these findings can be translated into clinical use [83].

**Atherosclerotic plaque inflammation**

**Author details**

**References**

Ida Gregersen\* and Bente Halvorsen

\*Address all correspondence to: ida.gregersen@rr-research.no

Inflammation is involved in all stages of plaque development. Endothelial dysfunction allows entry of lipoproteins and migration of inflammatory cells into the intimal layer of the artery. Inside the plaque the cells are activated by PAMPs and DAMPs. Cholesterol crystals are important DAMPs which can activate the NLRP3 inflammasome and stimulate release of inflammatory cytokines. Further, accumulation of large amounts of lipids in the immune cells can lead to extensive cell death, and a necrotic core develops due to dysfunctional clearance of these cells. The necrotic core maintains the nonresolving inflammatory milieu in the lesion and is a typical feature of advanced plaques. Moreover, tertiary lymphoid organs can form in the adventitial layer of the vessel wall and feed the plaque with inflammatory cells and mediators, and can further contribute to plaque inflammation. The authors wish to acknowledge Sverre Holm for making the illustration and SERVIER Medical Art (www.servier.fr) for use of their medical art kits.

Inflammatory Mechanisms in Atherosclerosis http://dx.doi.org/10.5772/intechopen.72222 39

Faculty of Medicine, Institute of Clinical Medicine, University of Oslo and Research Institute

[1] World Health Organization. Fact Sheet – Cardiovascular Diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs317/en/ [Assessed: October 8, 2017]

of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway

In sum, atherosclerosis is characterized by low-grade chronic inflammation in the arteries, in tight interplay with lipids. Targeting both pathways, depending on individual risk analysis, might be the future of prevention and treatment of cardiovascular disease. However, there is still a need for tools to identify people at risk, especially for personalized treatment. There is also a need to evolve more precise targets for treatment.

#### **Atherosclerotic plaque inflammation**

Inflammation is involved in all stages of plaque development. Endothelial dysfunction allows entry of lipoproteins and migration of inflammatory cells into the intimal layer of the artery. Inside the plaque the cells are activated by PAMPs and DAMPs. Cholesterol crystals are important DAMPs which can activate the NLRP3 inflammasome and stimulate release of inflammatory cytokines. Further, accumulation of large amounts of lipids in the immune cells can lead to extensive cell death, and a necrotic core develops due to dysfunctional clearance of these cells. The necrotic core maintains the nonresolving inflammatory milieu in the lesion and is a typical feature of advanced plaques. Moreover, tertiary lymphoid organs can form in the adventitial layer of the vessel wall and feed the plaque with inflammatory cells and mediators, and can further contribute to plaque inflammation. The authors wish to acknowledge Sverre Holm for making the illustration and SERVIER Medical Art (www.servier.fr) for use of their medical art kits.

#### **Author details**

capacity is however, as mentioned, compromised, leading to reduced clearance of dead cells, secondary necrosis and stimulation of the pro-inflammatory environment and growth of the necrotic core. SPMs are shown to counteract these processes and stabilize plaques [73, 77]. However, in advanced atherosclerotic lesions, the ratio of SPMs to pro-inflammatory mediators is decreased, and the administration of SPMs counteracts atherosclerotic disease development. These findings provide a mechanistic explanation for the defective resolution observed in atherosclerotic disease [78, 79]. To further map the production, regulation and function of pro-resolving mediators in atherosclerosis will be of great importance to increase our under-

Despite the great success of modern treatment modalities, atherosclerosis is still the leading cause of mortality and morbidity worldwide. For example, high-dose statin treatment and other standard measures only prevent a fraction of recurrent events in survivors of MI. This residual burden of events presents a pressing unmet medical need, and novel perspective on atherogenesis is needed to treat those who are not met by the current treatment regimes. An interesting new therapeutics is the enzyme proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors. PCSK9 binds to the hepatic low-density lipoprotein (LDL) receptor, thereby inhibiting recycling of this receptor, resulting in attenuated removal of LDL cholesterol (LDL-C) from the circulation. The importance of PCSK9 for LDL-C homeostasis is illustrated in individuals with loss- and gain-of function mutations in this enzyme leading to hypo- or hypercholesterolemia, respectively, with dramatic effects on the incidence of atherosclerotic disease. Recent studies have shown that anti-PCSK9 therapies markedly reduce LDL-C levels, leading to lower incidence of adverse cardiovascular disease (CVD) outcomes in high-risk patients with hyperlipidemia [80]. In patients with LDL levels that remain above the treatment target, despite statin treatment (residual LDL risk); adding a PCSK9 inhibitor should be considered. Recent network meta-analysis demonstrates that PCSK9 inhibitors significantly reduce LDL cholesterol, on top of medium to high statin therapy [81]. Of direct relevance for inflammation, a very recent study, the CANTOS trial, suggests that those with residual inflammatory risk could benefit from interleukin-1β inhibition by Canakinumab. This anti-inflammatory treatment resulted in reduced cardiovascular risk, independent of lipid-lowering effects [82]. Another exciting approach to target inflammation in atherosclerosis is the pro-resolving mediators SPMs. In contrast to anti-inflammatory agents, these ligands will not compromise host defense, one of the most important challenges of immunosuppressive therapeutics. Many experimental studies have shown therapeutic potential for SPMs; however, more knowledge is needed to pinpoint how these mediators act, before these

In sum, atherosclerosis is characterized by low-grade chronic inflammation in the arteries, in tight interplay with lipids. Targeting both pathways, depending on individual risk analysis, might be the future of prevention and treatment of cardiovascular disease. However, there is still a need for tools to identify people at risk, especially for personalized treatment. There is

standing of how the inflammation in atherosclerosis becomes nonresolving.

38 Atherosclerosis - Yesterday, Today and Tomorrow

**6. Inflammation: a therapeutic target in atherosclerotic disease**

findings can be translated into clinical use [83].

also a need to evolve more precise targets for treatment.

Ida Gregersen\* and Bente Halvorsen

\*Address all correspondence to: ida.gregersen@rr-research.no

Faculty of Medicine, Institute of Clinical Medicine, University of Oslo and Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway

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**Chapter 4**

**Provisional chapter**

**Effects of Nicotine Contained in Tobacco Mainstream**

**Effects of Nicotine Contained in Tobacco Mainstream** 

Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, the accumulation of vascular smooth muscle cells (VSMCs) have a phenotype differing from that of their normal contractile type. Nicotine is a major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. We investigated the changes in the expression levels of differentiation markers and activity of mitogen-activated protein kinases (MAPKs) after nicotine exposure for 48 h using human aorta primary smooth muscle cells (HVSMC) differentiated with transforming growth factor-β. The results indicated that HVSMC phenotype changed to a synthetic-like phenotype after nicotine exposure. Nicotine is a factor that can change the expression of differentiation marker proteins in VSMCs. Thus, we proposed that nicotine directly affects the migration of VSMCs from the tunica media to atheromatous plaques in the vascular intima by inducing the transformation from a contractile-type to a synthetic-like type, which occurs before the development of atheromatous plaques. Nicotine is contained in nicotine patches and gums for smoking cessation. There may also promote atheromatous plaque formation. We anticipate that determining this mechanism will lead to new means of preventing and treating plaque formation and development in arteriosclerosis.

**Keywords:** nicotine, vascular smooth muscle, cell migration, proliferation,

According to the World Health Organization (WHO) World Health Statistics 2016, the world's highest cigarette smoking rates were 76.2% for men in Indonesia, and 52.0% for women in Nauru. Ranked second place was Jordan for men (70.2%), and Kiribati for women (40.9%),

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.77010

**Smoke on Vascular Smooth Muscle Cells**

**Smoke on Vascular Smooth Muscle Cells**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77010

Akio Nakamura

Akio Nakamura

**Abstract**

cigarette smoke

**1. Introduction**

#### **Effects of Nicotine Contained in Tobacco Mainstream Smoke on Vascular Smooth Muscle Cells Effects of Nicotine Contained in Tobacco Mainstream Smoke on Vascular Smooth Muscle Cells**

DOI: 10.5772/intechopen.77010

#### Akio Nakamura Akio Nakamura

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77010

#### **Abstract**

Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, the accumulation of vascular smooth muscle cells (VSMCs) have a phenotype differing from that of their normal contractile type. Nicotine is a major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. We investigated the changes in the expression levels of differentiation markers and activity of mitogen-activated protein kinases (MAPKs) after nicotine exposure for 48 h using human aorta primary smooth muscle cells (HVSMC) differentiated with transforming growth factor-β. The results indicated that HVSMC phenotype changed to a synthetic-like phenotype after nicotine exposure. Nicotine is a factor that can change the expression of differentiation marker proteins in VSMCs. Thus, we proposed that nicotine directly affects the migration of VSMCs from the tunica media to atheromatous plaques in the vascular intima by inducing the transformation from a contractile-type to a synthetic-like type, which occurs before the development of atheromatous plaques. Nicotine is contained in nicotine patches and gums for smoking cessation. There may also promote atheromatous plaque formation. We anticipate that determining this mechanism will lead to new means of preventing and treating plaque formation and development in arteriosclerosis.

**Keywords:** nicotine, vascular smooth muscle, cell migration, proliferation, cigarette smoke
