Atherosclerosis

Chapter Atherosclerosis

Normal arterial vessel

Figure 1 normal arterial wall

Figure 2 summary of the comparison between normal and atherosclerotic arterial wall

Three layers of arterial vessel

The arterial vessel consists of intima, media and outer adventitia. (see Figure 1)

The intima is located closest to the arterial lumen and is therefore most ‘intimate’ with the blood. It is composed of a single layer of endothelial cells. These cells function as an active metabolic barrier between blood and the arterial wall.

The media is the middle layer and is the thickest of all. It is separated from the intima by internal elastic laminae and from the adventitia by external elastic laminae. The media consists of smooth muscle cells and extracellular matrix and play its role as a contractile and elastic of the vessel. The elastic function of the media is more distinguished in large arteries such as the aorta. It allows the vessel to stretch during systole and then to contract during diastole in order to pump the blood forward. In smaller arteries such as arterioles, the muscular component is more prominent. The muscle cells act as a constrictor or relaxer of the vessel in order to alter luminal blood flow by influencing the resistance of the vessel.

The outer adventitia provides nourishment to the cells of the vessel by means of nerves, lymphatics and vasa vasorum.

Arterial wall is constantly concerned with dynamic interchange between its cellular components and their surrounding extra cellular matrix. By understanding the physiology of this dynamic interchange and the functions of each cellular component the dysfunction of these cellular components leading to atherogenesis are understood. (see Figure 2)

Role of cellular components in atherogenesis

Endothelial cells

Normal artery wall contains endothelial cells that manage the homeostasis of the wall by structural, metabolic, and signaling functions. The endothelial cells are tightly joined with each other in order to form a suitable barrier that keeps the blood inside the vessel and inhibits the large molecules to pass from the blood to subintima (subendothelial space). It is thus an active biologic interface between the blood and other tissues. Endothelium has several important functions such as regulation of thrombosis, contraction of smooth muscle cells of the vessel, and immune response.

The endothelium produces antithrombotic molecules in order to prevent blood from clotting. Certain molecules such as heparin sulfate, thrombomodulin, and plasminogen dwell on the endothelial surface and other molecules such as prostacyclin and nitroic oxide (NO) enter the blood. Endothelium can produce prothrombotic molecules when it encounters various stressors, however normally it maintains a net anticoagulant state.

Another function of endothelium is to modulate contraction of smooth muscle cells in the media by releasing substances such as vasodilators and vasoconstrictors. Vasodilators (e.g. NO, prostacyclin) and vasoconstrictors (e.g. endothelin) fine-tune the resistance of the vessel and subsequently alter the arterial blood flow. Endothelium normally maintains a state of net smooth muscle relaxation with the predominance of vasodilators.

Endothelial cells have an important function as a regulator of the immune response. In a normal situation without the pathologic stimulation, endothelial cells work as anti-inflammatory by resisting leukocyte adhesion. When local injury or infection initiates pathologic stimulation, endothelial cells respond by secreting chemokines that attract white blood cells to the injured area. Additionally, endothelium produces cell surface adhesion molecules, which hold mononuclear cells to the endothelium, and therefore promote their migration to the injury site.

In conclusion, the normal arterial endothelium implements good barrier with net anticoagulant properties, net relaxation of the smooth muscle and anti-inflammatory trait.

Vascular smooth muscle cells

Smooth muscle cells have two important functions; contractile and synthetic. The contractile function of these cells are stimulated or inhibited by various vasoactive substances such as angiotensin II, acetylcholine, endothelin, and NO. Such modulation results in vasoconstriction or vasodilatation.

Synthetic function of smooth muscle cells is important to understand, because it may contribute to the pathogenesis of atherosclerosis. Normally they synthesize collagen, elastin, and proteoglycans that form the vascular extracellular matrix. They also provide vasoactive and inflammatory mediators, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α). These mediators stimulate leukocyte proliferation and induce the endothelial cells to express leukocyte adhesion molecules as mentioned earlier.

Extracellular matrix

Vascular extracellular matrix in the media consists of elastin, proteoglycans and fibrillar collagen. With the provision of flexibility by elastin, and biomechanical strength by fibrillar collagen, the arterial vessel is able to maintain the structural integrity despite the high pressure within the lumen. Fibrillar collagen can also inhibit the proliferation of smooth muscle cell in vitro according to recent evidence. The matrix also plays a role in the process of cellular responses to stimuli, such as growth factors and may for example deter the cells to undergo apoptosis.

Atherosclerotic arterial vessel

Figure 3 summary of 3 pathologic stages of atherosclerosis

Three pathologic stages of atherogenesis

Endothelial cells and smooth muscle cells may respond to inflammatory mediators when normal homeostasis is disrupted. Recent research has shown that inflammatory mediators such as IL-1 and TNF-α can multiply themselves by activating vascular cells to produce such cytokines. Thus this research pointed out that not only the immune cells, but also the “activated” vascular cells can be proinflammatory. As the process of atherogenesis is believed to be caused by proinflammatory agents, it is important to discover this proinflammatory trait of vascular cells. This research also identified several other factors that contribute to atherogenesis , such as endothelial dysfunction, formation of lipid layer within the intima, migration of leukocytes and smooth muscle cells to the vessel wall, formation of foam cells, and deposition of extracellular matrix. Formation of the plaque is a chronic process of continuous interaction and competition among the cells of the lesion. This process can be identified in three stages; the fatty streak, plaque progression, and plaque disruption. (see Figure 3)

Fatty streak

The earliest visible sign of atherogenesis is the fatty streak, which means areas of yellow discoloration on the surface of the artery lumen. At this stage, this fatty streak doesn’t protrude substantially into the artery wall nor impede blood flow. This process is already visible in most people by the age of 20. There are no symptoms and this lesion may even diminish over time. Research based on several animal models suggest that various stressors such as physical forces and chemical irritants cause early endothelial dysfunction, which allows entry and modification of lipids within the subintima. This modified layer of lipids serve as proinflammatory mediators and thus initiates the migration of leukocytes and formation of foam cells.

The distinctive hallmark of the fatty streak is the initiation of migration of leukocytes and formal cell formation caused by proinflammatory mediators.

Endothelial dysfunction

Endothelial dysfunction is a primary event in atherogenesis, which can be caused by various agents, such as physical stress and chemical irritants. Observations showed that atherosclerosis often forms at arterial branch points, for example at bifurcations and this result proposes that physical stress plays an important role in atherogenesis. There are several projective mechanisms against atherosclerosis by endothelial cells. One of them is NO, which is an endogenous vasodilator that works as an inhibitor of platelet aggregation and as an anti-inflammatory product. It is secreted by endothelial cells when they are stimulated by laminar flow in erect sections of arteries. Another mechanism is the expression of the antioxidant enzyme superoxide dismutase by the endothelium. This enzyme works against reactive oxygen species, produced by chemical irritants or transient ischemia in the vessel.

Unfortunately, these atheroprotective endothelial functions can be impaired by several factors. For example, disturbed flow (physical stressor), typically located at arterial branch points, can impair the protective functions. This is well illustrated by the difference in prevalence of atherosclerosis deposition between branched arteries and bifurcated vessels. Bifurcation areas such as the common carotid and left coronary arteries are relatively more common deposition sites for atherosclerosis than arteries with few branches such as the internal mammary artery.

Chemical irritants such as cigarette smoking, abnormally high circulating lipid levels or high sugar levels (diabetes mellitus) can contribute to endothelial dysfunction and are all well- known risk factors for atherosclerosis. Exposure to chemical irritants promotes endothelial dysfunction by increasing endothelial production of reactive oxygen species, which alter the metabolic and synthetic functions of endothelial cells. As a result, the endothelium is inclined to exhibit proinflammatory processes.

Hemodynamic and chemical stressors contribute to distorting the endothelial homeostasis and promote endothelial dysfunction. The following undesired effects result from; impairment of permeability barrier function, secretion of inflammatory cytokines, stimulation of adhesion molecules on the cell surface that promote leukocyte recruitment, and altered antithrombotic properties and release of vasoactive molecules. Consequently, these undesired effects establish the groundwork for further advancement of atherosclerosis.

Lipoprotein entry and modification

Impairment of permeability barrier due to endothelial dysfunction allows the passage of circulating lipoproteins (low-density lipoprotein, LDL) into the intima. By binding to the extracellular matrix component called proteoglycans, LDL assures its place in intima and starts accumulating. This accumulation is a critical process in atherogenesis since LDL may undergo chemical modifications while residing longer in intima. It is needless to say that an elevated circulating LDL concentration strongly contributes to this accumulating process. Another major risk factor for this process is hypertension. High blood pressure influences smooth muscle cells to promote LDL-binding with proteoglycans and therefore also contributes to “trapping” of lipoproteins within the vessel wall.

As mentioned earlier, chemical modification occurs with LDL when chronic accumulation takes place inside intima. There are several types of chemical modification that may occur. One is called oxidation and it results from the chemical reaction of reactive oxygen species and pro-oxidant enzymes produced by endothelial or smooth muscle cells, or macrophages penetrating the intima. Diabetes is a major risk factor for atherosclerosis since chronic hyperglycemia can stimulate glycation of LDL that may ultimately alter LDL into an antigenic and proinflammatory molecule. In conclusion, this biochemical modification of LDL into proinflammatory molecule does not only contribute to inflammation by endothelial dysfunction, but it also maintains the promotion of leukocyte recruitment and foam cell formation throughout the plaque development.

Leukocyte recruitment

Leukocyte recruitment to the arterial wall is another key step in atherogensis, which is dependent on two important factors; expression of leukocyte adhesion molecules (LAM) on endothelial wall, and chemoattractant signals such as IL-8 that direct diapedesis (intruding of molecules through the intact vessel wall). These two factors mainly direct monocytes to the atherosclerotic lesion although T lymphocytes play the central role in the immune system. T-lymphocyte resides within plaques at all stages of atherogenesis, mainly producing cytokines.

As mentioned earlier, modified LDL can maintain leukocyte recruitment by inducing LAM and chemokine expression. It can also stimulate endothelial and smooth muscle cells to produce proinflammatory cytokines such as IL-1 and TNF-α. These proinflammatory cytokines can also induce LAM and chemoattractant cytokine expression equivalent to modified LDL. Therfore, the dual ability of modified LDL can directly or indirectly promote leukocyte recruitment and throughout atherogenesis.

Foam cell formation

When monocytes enter the intima, they differentiate into phagocytic macrophages. These phagocytic macrophages may become foam cells when they absorb lipoproteins. They don’t uptake LDL from a classic cell surface LDL-receptor, since it does not recognize modified LDL, but from a family of ‘scavenger’ receptors that do bind and internalize modified LDL. Uptake by scavenger receptors avoids negative feedback inhibition from the high cholesterol content unlike the classic LDL-receptors, and allows the macrophages to imbibe cholesterol-rich lipid that results into the formation of foam cells. This uptake seems to be beneficial at first sight, since it absorbs the inflammatory modified-LDL, however when these foam cells have impaired outflow, they will be locally accumulated in the plaque and encourage the plaque progression by serving as a source of proinflammatory cytokines.

Plaque progression

Smooth muscle cells are the ones who play a central role in plaque progression. Atherosclerotic plaque has two typical features, which are thrombogenic lipid core and a protective fibrous cap. Plaque growth does not always cause luminal restriction of blood flow at an early stage thanks to a compensatory outward remodeling of the plaque wall. This remodeling preserves the diameter of the vessel lumen and thus may even evade detection by angiography. Nevertheless, plaque growth at a later stage may result in ischemia due to the narrowing of the vessel and can cause ischemic symptoms such as angina pectoris or intermittent claudication.

Smooth muscle cell migration

Migration of smooth muscle cell from the media to the intima is a distinctive indication of transition from fatty streak to plaque formation. After the migration, smooth muscle cells proliferate within the intima and secrete extracellular matrix macromolecules.

Additionally, foam cells, activated platelets, and endothelium stimulate the substances that signal the migration and proliferation of smooth muscle cell. For example, foam cells release platelet derived growth factor (PDGF), cytokines and growth factors that directly contribute to the migration and proliferation process, and they also activate smooth muscle cell and leukocyte to reinforce inflammation in the atherosclerotic lesion.

Although plaque progression is traditionally known as a gradual and continuous process, recent evidence claim that this process can be strongly accentuated by bursts of smooth muscle replication. The observation of small ruptures within the plaque occurring without any clinical symptoms or signs supports this suggestion. These small ruptures expose tissue factor secreted by foam cells that stimulates coagulation and microthrombus formation in the lesion. Such microthrombus contains activated platelets that release additional factors such as PDGF and heparinase that can further stimulate local smooth muscle cell migration and proliferation. Heparinase stimulate smooth muscle cell migration and proliferation by degrading heparin sulfate, which normally counteracts this process.

Extracellular matrix metabolism

Metabolic process in extracellular matrix plays a central role in bridging the plaque progression to plaque rupture. Ultimately, this process weakens the fibrous cap, predisposing it to rupture. This process is influenced by the balance of matrix deposition synthesis by smooth muscle cells and degradation by matrix metalloproteinases (MMP), a class of proteolytic enzymes. For example, PDGF and TGF-β stimulate interstitial collagen production, while inflammatory cytokines such as IFN-γ inhibits collagen synthesis. Inflammatory cytokines also weakens the fibrous cap by stimulating local foam cells to secrete MMP that degrades collagen and elastin of the fibrous cap.

Plaque disruption

Plaque integrity

Chronic shifting of the balance of extracellular matrix metabolism leads to serious consequences to the plaque integrity. As mentioned earlier, it accelerates inflammatory stimulation or activation of apoptosis pathways and therefore leads to death of smooth muscle and foam cells. Cell death leads to release of cellular contents, whereby more lipids and cellular debris is absorbed to the dynamic lipid core. This process increases the size of the lipid core and as a result alters biomechanical environment and hence the stability of the plaque. Plaque border adjacent to the normal tissue is called shoulder region and it’s the main location where the hemodynamic stress is focused. As the size and the protrusion of the plaque in the vessel increase, the hemodynamic stress will also increase around the shoulder region. Furthermore, local accumulation of foam cells and lymphocytes at this site makes the plaque more susceptible to rupture by accelerating degradation of extracellular matrix.

Plaque integrity and its vulnerability to rupture are highly dependent on the net balance of deposition and distribution of the fibrous cap. When the fibrous cap is very thick, the plaque is called stable plaque and it may reinforce the narrowing of the artery, but on the other hand diminish the susceptibility to rupture. Plaques with thinner fibrous caps are called vulnerable plaques and they cause less obstruction in the artery, but are more fragile and have higher susceptibility to rupture and trigger thrombosis. Characteristically, stable plaques have thick fibrous cap with small lipid core, while vulnerable plaques have thin fibrous cap with rich lipid core, extensive macrophage infiltrate and weakening smooth muscle cells. It is difficult to foresee the ‘clinical’ consequences of the plaque.

Thrombogenic potential

Disruption of the fibrous cap does not always lead to major clinical events such as myocardial infarction and stroke. For example, small non-occlusive thrombi may be reabsorbed into the plaque, continuing the process of smooth cell growth and fibrous deposition. The extent of how occlusive and transient the thrombus will be is largely dependent on the thrombogenic potential of the plaque.

The counter balancing of coagulation and fibrinolysis determines the probability of a major clinical event due to occlusive thrombosis. Inflammatory stimuli in the plaque environment incite smooth muscle cells, endothelial cells, and foam cells to release tissue factor that initiates the extrinsic coagulation pathway. Inflammatory stimuli also stimulate expression of antifibrinolytics such as plasminogen activator inhibitor-1 and consequently enhance thrombosis. As mentioned earlier, the activated endothelial cells also contribute to thrombosis and coagulation by depositing fibrin at the vascular wall.

The concept of ‘vulnerable plaque’ has developed into a new concept of ‘vulnerable patient’ as the recent evidence shows that a person’s susceptibility to coagulation and thus vascular events can be influenced by many other factors such as genetics (e.g. procoagulant prothombin gene mutation), coexisting condition (e.g. diabetes), and lifestyle factors (e.g. smoking, obsesity).

1.3 Complications of atherosclerosis

Developing of atherosclerotic plaques is not homogenous throughout the vasculature. The following anatomical structures are the most common areas where atherosclerosis takes place, starting from the most common region: dorsal section of the abdominal aorta, proximal coronary arteries, the popliteal arteries, descending thoracic aorta, internal carotid arteries, and renal arteries. It is needless to say that the areas perfused by these vessels are most frequently impaired by the consequences of atherosclerosis. Clinically, this can lead to thromboembolism resulting in major cardiovascular diseases such as stroke and myocardial infarction.