**3. Atherosclerosis**

Atherosclerosis can be defined as a disease that causes progressive arterial stenosis and obstruction due to intimal plaques containing lipids, fibroblasts, macrophages, smooth muscle cells, and extracellular substances in varying proportions, leading to loss of the elasticity and antithrombotic properties of the arterial walls. Atherosclerosis is a multifactorial, morbid and mortal systemic disease that affects not only the coronary vessels but all arterial structures. The coronary arteries, internal carotid arteries, and abdominal aorta are vessels most commonly affected [42].

Atherosclerotic lesions appear as focal thickenings in the intima and subintimal space of arteries. When the content of the thickened region is examined, it is seen to contain vascular endothelial cells, smooth muscle cells, connective tissue, and lipid deposits, as well as inflammatory and immune cells from the blood. Historically, with our nascent understanding of atherosclerosis, treatment focused on cholesterollowering drugs and a low-cholesterol diet. However, research on therapeutic processes started to diversify after the role of inflammation in atherosclerosis came to light.

#### **3.1 Atherosclerosis and inflammation**

Atherosclerotic vascular disease is a typical environment-gene interaction. Environmental risk factors trigger a proinflammatory response in people with genetic predisposition. Epidemiological studies have demonstrated the role of risk factors such as cigarette exposure, cholesterol, hypertension, and diabetes mellitus in the development of atherosclerosis. Experimental studies have shown that these risk factors induce a general inflammatory response, causing a widespread reaction in the body. In response to risk factors, systemic acute phase reactants are activated and there is the onset of signal traffic from the endothelium. In light of this information, atherosclerosis is defined as a multifactorial disease that is associated with the inflammatory process and in which chronic inflammation plays a role in every stage, from onset to progression [43–45].

At the onset of atherosclerosis, leukocytes and macrophages attach to the endothelium and cross into the subendothelial space. This occurs because of adhesion molecules [46, 47]. The best-known adhesion molecules are endothelial leukocyte adhesion molecule 1 (ELAM-1), membrane-bound vascular cell adhesion molecule 1 (VCAM-1), and intracellular adhesion molecule 1 (ICAM-1) [48, 49].

Cytokines are also an integral factor in the inflammatory process. The best-known proinflammatory cytokines are tumor necrosis factor alpha (TNF-ɑ), interleukin (IL)-1, and IL-6. TNF-ɑ is released from macrophages, vascular smooth muscle cells, and endothelial cells. These cytokines trigger the production of other cytokines in the inflammatory cycle [50].

Another marker is plasma fibrinogen values, which are an indicator of both the inflammatory response and the thrombotic response. Increased fibrinogen values have been shown to significantly increase coronary event risk. In addition, elevated fibrinogen levels have been detected in healthy individuals with a family history of atherosclerosis [51, 52].

C-reactive protein (CRP) is a good indicator of inflammation because its values are stable over time [53, 54]. It does not increase due to anything other than inflammation. It can be measured with a highly sensitive and inexpensive test. Studies have shown that CRP levels have an additive effect on other risk markers. The cholesterol/ high-density lipoprotein (HDL) ratio is a strong indicator of cardiovascular risk [55]. However, the risk predictivity increases when CRP values are added. The PROVE-IT study showed that highest risk group is those with both high total cholesterol/HDL ratio and high CRP levels [56].

Elevated CRP levels, increased leukocyte counts in peripheral blood counts, and high serum fibrinogen levels are strong predictors of coronary artery disease and atherosclerotic diseases [57, 58].

The normal arterial structure consists of three main layers, the intima, media, and adventitia from innermost to outermost. The intima layer is covered with a single cell layer endothelium. The intact endothelial surface is resistant to thrombus formation because it secretes nitric oxide (NO) and prostacyclin (PGI2) and is covered with heparin sulfate.

#### **3.2 Endothelial inflammation**

The endothelium is the first vascular structure affected by risk factors [59, 60]. Normally shiny, slippery, and antithrombotic, risk factors cause the endothelium to lose its slipperiness and become sticky and prothrombotic. Endothelial cells exposed to risk factors from an early age start producing adhesion molecules (VCAM-1, ICAM), growth factors (platelet-derived growth factor [PDGF], basic fibroblast growth factor [FGF], *transforming growth factor beta* [TGF-β], IL-1, TNF-α), and cytokines (macrophage colony-stimulating factor [M-CSF], granulocyte-macrophage colony stimulating factor [GM-CSF]). VCAM-1 binds both monocytes and T lymphocytes. Atherosclerosis-related leukocyte adhesion molecule, or athero-ELAM, is released from endothelial cells, triggering mononuclear cell migration [61]. This initiates the chemotactic process on monocytes, macrophages, and lymphocytes and triggers the inflammation process [62]. The result is a vicious cycle in which inflammation stimulates cytokine release and cytokines increase inflammation. These proteins, which are expressed due to the vascular inflammatory response, are the main cause of early atherosclerotic lesions [62].

On the one hand, there is an inflammatory response in the endothelium, while on the other hand, there is subclinical systemic inflammation. Proinflammatory risk factors such as oxidized low-density lipoprotein (LDL) activate IL-1 and TNF-α, which are called primary proinflammatory cytokines [53, 63]. These primary proinflammatory cytokines activate IL-6, resulting in the release of acute phase reactants. The presence of subclinical systemic inflammation can be understood by measuring some acute phase reactants such as CRP, fibrinogen, factor 7, plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and lipoprotein (a) in the blood or by measuring endothelium-derived peripheral markers [53, 64, 65].

### **3.3 Medial inflammation**

In the atherosclerosis process, smooth muscle cells migrate from the media to the intima and there is a reduction in the contractile protein content and an increase in the number of synthetic organelles. Smooth muscle cells migrating to intima change from the contractile phenotype to the synthetic phenotype and contribute to proliferation. Smooth muscle cells in the media respond to vasoconstrictors such as endothelin, catecholamine, angiotensin II, and vasodilators such as NO and PGI2, while those in the intima respond to mitogens such as PDGF. In addition, the balance shifts from vasodilation to vasoconstriction, from antithrombotic to prothrombotic, and from antiproliferative to proliferative properties. Adhesion molecules, cytokines (IL-1, TNF-α), chemokines (monocyte chemoattractant protein-1 [MCP-1], IL-8), and growth factors (PDGF, FGF) are released from dysfunctional endothelial cells. IL-8 triggers the inflammatory cascade by binding to chemokine receptor 2 on leukocytes [66]. MCP-1 mediates selective directed migration of monocytes to the subendothelial space. Transgenic experimental animals unable to express MCP-1 were found to have nearly absent subendothelial lipid accumulation [67] All these processes allow defense cells to migrate to the inflammation site, leading to the onset of volumetric thickening of the vessel wall.

### **3.4 Lipid deposition and atherosclerosis**

Lipids are a key cell component that serves as one of the main building blocks of cell membranes and organelles, as well as having nutrient and energy functions. Fatty acids, the simplest lipid form, are divided into different classes depending on the length of their structure, the number of carbon atoms, and whether the bonds are saturated or unsaturated. Phosphoglycerides are the main class of lipids comprising cell membranes. Cholesterols are also part of a large group of fats called sterols and are another important component of cellular membranes. LDL particles in the blood are made of lipids and protein, including cholesterol esters, triglycerides, phospholipids, and apoB-100 protein.

Many studies indicate that atherosclerosis begins with endothelial damage. However, histopathological studies have revealed atherosclerotic plaque formations with an intact endothelial structure [68, 69]. This raises the question of how lipid and cell passage into the subendothelial space occurs without endothelial damage.

It has been observed that eating even a single meal of excessively fatty food disrupts endothelial function, raises CRP levels, and increases adhesion molecules [70]. Animal experiments have shown that in subjects fed a high-cholesterol diet, the endothelium becomes sticky and begins expressing adhesion molecules within a few weeks [71–73]. The first alteration in the arterial endothelium of experimental animals fed a cholesterol-rich diet was shown to be leukocyte adhesion [74].

The main damage caused by cholesterol particles occurs through LDL. LDL particles are believed to penetrate the arterial wall by passing through the endothelial cells and initiate a number of remarkable changes involving various different processes. Subintimal lipid particles have been shown to be ingested by macrophages and smooth muscle cells, where they are degraded in the intracellular lipid oxidation and peroxidation chains. In the sub intimal space, LDL particles are modified with a different phospholipid and fatty ester structure and begin to form lipid clusters [75]. These lipid clusters trigger free radicals produced by chain chemical reactions, induce the inflammatory process, and cause chemotaxis.

#### **3.5 Fibrous plaque–fibrous cap**

Although immunohistochemical studies have demonstrated a cascade mechanism that allows inflammatory cells to infiltrate the subintimal layer at this early stage of atherosclerosis, pathological studies show that the endothelium is intact at this stage and there is no physical damage to this layer on microscopic examination [68]. Light microscopy examination of very small early lesions showed that primary damage occurred in the muscle cell component of the intima.

Monocytes accumulated in the subendothelial space transform into macrophages and begin to express scavenger receptors. This enables them to phagocytose the oxidized LDL.

As cholesterol esters accumulate in the macrophages, foam cells are formed. Macrophages accumulate lipids while continuing to release inflammatory mediators. M-CSF released from activated endothelial cells increases macrophage accumulation in the region. M-CSF also stimulates the immune system. A proinflammatory cytokine called CD40 ligand is one of the inflammatory mediators that contribute to progression. T cells accumulate in the subendothelial space due to the effect of different chemokines (e.g., interferon gamma-induced protein 10 [IP-10], monokine induced by interferon gamma [MIG]). Mast cells have recently been shown to accumulate via similar mechanisms. T lymphocytes also accumulate in the intima and continue to release proinflammatory cytokines. Another interesting function of T cells is to activate macrophages to stimulate the release of collagen, matrix metalloproteinases (MMP), and cytokines. Thus, the atheromatous plaque gradually grows. Oxidized LDL and heat shock protein (HSP) increase inflammation by stimulating toll-like receptors [76, 77]. Experimental studies have shown that toll-like receptor blockade can reduce atherosclerosis. Toll-like receptors accelerate atherosclerosis by triggering cytokine release in the inflammation cascade and stimulating the immune response [78].

As the lesions progress, extracellular lipids begin to accumulate. The extracellular lipid pool is largely a result of foam cell apoptosis and the release of their stored cholesterol esters. A very small proportion comprises lipoproteins that pass from the lumen. The "fibrous plaque" that begins to form in the subintimal layer initially appears microscopically as lipid nuclei, large amounts of smooth muscle cells, macrophages, foam cells, T lymphocytes, and extracellular matrix, and macroscopically as white lesions that enlarge mostly towards the artery lumen [3].

Smooth muscle cells in the fibrous plaque continue to produce extracellular matrix, while macrophages degrade the connective tissue. This construction and destruction is mediated by numerous cytokines. Even if fibrous plaques significantly narrow the vessel lumen, they are believed not to cause significant clinical events as long as they remain intact. The structure on the luminal side of this plaque is called the "fibrous cap." A thicker fibrous cap is associated with greater plaque stability.

Plaques that are rich in lipids and inflammatory cells and have a thin fibrous cap have higher risk of rupture (vulnerable plaque). Metalloproteinases (collagenase, elastase, stromelysin) secreted by macrophages surrounding the lipid nucleus degrade the collagenous matrix of the fibrous cap. In addition, the synergistic effect of IL-1β and TNF-α released from activated macrophages and interferon gamma (IFN-γ) released by T lymphocytes results in smooth muscle cell death and reduced extracellular matrix. As a result of increased destruction and decreased construction, the fibrous cap weakens and eventually ruptures. Procoagulant substances in plaques with a disrupted fibrous cap interact with blood elements and clotting factors, triggering thrombus formation [65].

*A Hidden Organism, Chlamydia in the Age of Atherosclerosis DOI: http://dx.doi.org/10.5772/intechopen.109745*

The ruptured plaque remains unstable for some time, after which the healing process begins. Smooth muscle cells capable of making extracellular matrix act as reparative cells. Smooth muscle cells produce large amounts of matrix proteins, such as glycosaminoglycan, elastin, and collagen, which are needed to repair the vessel and form the fibrous cap over the lipid-rich plaque nucleus. By synthesizing its contents, they enable the plaque capsule to stabilize the atherosclerotic lesion and separate the thrombogenic lipid-rich plaque nucleus from the platelets and coagulation cascade proteins in the blood. Thus, vascular smooth muscle cells have a critical role in ensuring plaque stability and inhibiting fatal thrombogenic outcomes. Some authors have argued that smooth muscle cells migrating into the intima play a constructive and reparative role, rather than a destructive role, in atherosclerosis [45, 79].

#### **3.6 Atherosclerosis and immune response**

It has recently become understood that both the natural and adaptive immune systems play important roles in the development of atherosclerosis [80, 81]. The natural immune system is responsible for the initial inflammatory response to a microorganism or pathogen. Immune cells, namely T cells, monocytes, macrophages and mast cells, circulating through various tissues (including the atherosclerotic artery) seeking antigen. When T cells encounter and bind to an antigen, a series of cytokines are released to launch an inflammatory response. Scavenger and toll-like receptors are the main receptors responsible for natural immunity in atherothrombosis [82]. Tolllike receptors are found on fibroblasts and macrophages in the intimal and adventitial layers of coronary atherothrombotic plaques.

The adaptive immune system is more specific than the natural immune system. This system includes an organized immune response leading to the formation of T and B cell receptors and immunoglobulins that recognize foreign antigens. Modified lipoproteins, HSPs, beta2-glycoprotein I, and infectious agents can stimulate the adaptive immune system [83].
