Pathophysiology of Atherosclerosis

Introduction

Credited to Felix Marchand in a 1904 publication, the name atherosclerosis roughly translates as ‘hardened gruel,’ This colorful term, reminiscent of other culinary metaphors in pathology, underlies many of the most feared and lethal conditions in medicine. Atherosclerosis is primarily an arterial disorder, classically characterized by lipid deposition in the vessel intima, and associated with inflammation, scarring, and calcification. Eventually these lesions cause luminal stenosis and potentially culminate in thrombotic occlusion and/or embolism. Atherosclerosis is highly prevalent in the industrialized countries, with a growing frequency in all geographic regions of the world. Risk factors include elevated serum cholesterol, diabetes mellitus, smoking, male gender, advancing age, obesity, systemic chronic inflammation, and as yet incompletely defined genetic factors. Since the manifestations of atherosclerosis – including coronary artery disease (CAD), peripheral vascular disease (PVD), abdominal aortic aneurysm (AAA), renal artery stenosis (RAS), and carotid artery stenosis (CAS) – account for a significant fraction of worldwide morbidity and mortality, substantial effort has been expended to understand its pathogenesis. We have done well in identifying – and modifying – risk factors for atherosclerosis; modern medical intervention also does a credible job in treating the sequelae of this ‘hardened gruel’. However, there is much that needs to be learned and a better understanding of the basic cellular mechanisms will allow us the opportunity to potentially prevent atherosclerosis before it even begins.

Atherosclerosis often begins relatively early in life. Autopsy and radiologic studies have revealed that even teenagers can have atherosclerotic plaque in their aortas and coronary arteries. At the other end of the spectrum, older individuals may have arteries so calcified by this process that they appear similar to bone on routine radiologic imaging.

Pathologically, atherosclerotic lesions can appear very similar at different sites throughout the body. However, there is marked heterogeneity in the manner in which patients with atherosclerosis present with clinical symptoms. The inflammatory component of atherosclerosis can damage vessel walls to the extent that they can weaken and dilate, leading to aneurysm formation. In renal arteries, atherosclerotic occlusion can restrict kidney perfusion, leading to compensatory activation of the renin–angiotensin axis and poorly controlled systemic hypertension. Arteries in the legs may become so stenotic as to no longer serve the limb sufficiently, causing distal limb ischemia, and necessitating surgical vascular bypass operation or even amputation. Lesions in the carotid arteries may lead to ischemic or embolic stroke. In the heart, atherosclerosis can either slowly occlude arteries–leading to angina, or can suddenly rupture–causing downstream infarction or even sudden death.

Individuals can present with preferential involvement of one vascular site over another. Thus, some patients present primarily with carotid artery stenosis and stroke, while others present primarily with coronary artery disease and myocardial infarction. Similarly, one patient may have severe coronary artery disease and only mild aortic atherosclerosis, while another patient may have severe aortic atherosclerosis and only mild coronary artery involvement. It is likewise unclear why some branching arteries such as the internal mammary artery are relatively protected from atherosclerotic involvement, while other branching arteries, such as the carotid and coronary arteries, are considerably more susceptible. These observations highlight the fact that there is much we still do not understand.

Early Lesions

The earliest visualizable lesion of atherosclerosis is the fatty streak, which is an accumulation of lipid-laden macrophages in the vascular intima ( Figures 12.1, 12.2 ). Fatty streaks can be appreciated grossly as focal yellow areas of discoloration of intimal surface. These lipid-laden macrophages are often referred to as foam cells because of their foamy appearance. These foam cells are believed to derive from the ingestion of lipids by ma- crophages within the intima. Research into the mechanisms leading to the formation of early atherosclerotic lesions is ongoing, but clearly a number of factors play important roles. In the current understanding of atherogenesis, the three most important factors, all interacting with each other, appear to be altered lipid metabolism, vascular cell activation, and inflammation.

Lipids in Atherosclerosis

Accumulation of lipid in an interstitial space obviously requires a source of lipid. Thus circulating lipid, ostensibly to fulfill essential biologic functions, becomes trapped in the tissue of the blood vessel without ever reaching the intended target cells. This accumulation appears to be promoted in the context of increased levels of lipids in the bloodstream, and in particular, increased levels of cholesterol. Cholesterol circulates in the blood as multiple distinct forms of lipoprotein particles. In particular, low-density lipoprotein (LDL)-associated cholesterol (so-called bad cholesterol) promotes atherosclerosis development. In contrast, high-density lipoprotein (HDL) particles (so-called good cholesterol) promote reverse cholesterol transport, removing cholesterol from the vessel wall. LDL cholesterol can thus be viewed as the fuel for the fire of atherosclerosis. There is no one critical threshold level of LDL that will trigger development of atherosclerosis, and since all humans have circulating LDL, all who live until adulthood will develop atherosclerosis to some extent. In most people, the atherosclerosis remains mild and subclinical, until late in life. However, the degree to which the atherosclerotic lesions in any one person will progress is impacted by the concentration of LDL in the blood of that person.

An important observation in the field of atherosclerosis research is that LDL particles that have entered the vessel wall are often chemically modified prior to being engulfed by macrophages. Thus LDL can undergo oxidative modifications (oxidized LDL) by the reactive oxygen species generated in the vessel wall at sites of vascular cell activation and inflammation. LDL may also undergo non-enzymatic glycation, particularly in patients with diabetes. Such modifications of LDL can facilitate its entrapment in the intima by altering its interaction with the extracellular matrix. More importantly, these modifications greatly enhance the degree to which macrophages will ingest the LDL particles. Macrophages can ingest modified LDL using specialized receptors called scavenger receptors. These receptors are distinct from the traditional LDL receptors used by the liver and other tissues to internalize normal LDL present in the serum.

Genetic variations in genes encoding proteins that control the metabolism of cholesterol and lipids can markedly impact a person’s propensity to develop atherosclerosis. Such genes include those coding for the LDL receptor (LDLR), apolipoprotein(a) (apo(a)) and apolipoprotein E (ApoE) (see Genetics of Atherosclerosis, below). The primary deleterious impact of many of these pro-atherogenic genetic variations is an increase in the levels of circulating lipoproteins. These findings in people have led to the development of genetically engineered mouse strains that develop marked hyperlipidemia and atherosclerosis, and are widely used to study the pathogenesis of atherosclerosis (see below).

Endothelial Activation

Atherosclerosis results in large part from the interaction of lipoproteins with the vessel wall. Thus, changes in the cells of the vessel wall itself play an important role in the formation of atherosclerotic lesions. Activation of endothelial cells and vascular smooth muscle cells is a relatively general response of these cells to diverse stimuli (see Chapter 11 ). Activation of vascular cells is also an important component of atherogenesis. Since the endothelium sits at the border between the circulating blood and the vessel wall, it plays particularly important roles in this disease. In fact, phenotypic changes in endothelial cells impact the development of atherosclerosis in several distinct ways.

It is the endothelium that largely determines the specific section of an artery that will first develop atherosclerosis. Endothelial cells are responsive to biomechanical stimuli imparted by blood flow. Steady laminar shear stress promotes an ‘atheroprotective state’ in endothelial cells. However, disturbed blood flow, i.e. near branch sites in the vasculature, causes endothelial changes that promote atherosclerosis.

Endothelium that experiences laminar shear stress within the blood stream promotes vasodilation and is anti-thrombotic. However, activated endothelial cells become less efficient in promoting vasodilation, in preventing platelet aggregation, and in suppressing coagulation. The shift toward such endothelial dysfunction is associated with increased reactive oxygen species production (e.g., superoxide and hydrogen peroxide), and a decreased generation of nitric oxide. This flow-related, site-selective activation of endothelial cells is believed to underlie the observation that atherosclerosis develops first and most severely at sites near vascular bifurcations, including the proximal left anterior descending artery of the heart, just after its bifurcation from the left main coronary artery.

The activated pro-atherogenic endothelium facilitates increased lipid uptake into the vessel wall through increased metabolism and altered transendothelial transport. Additionally, the activated endothelium expresses surface adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-selectin, and E-selectin. These adhesion molecules bind circulating inflammatory cells in the blood in order to recruit them to the site of vascular activation. The activated endothelial cells also secrete cytokines and chemokines that attract and activate these inflammatory cells. The activated endothelium secretes large amounts of growth factors, such as platelet-derived growth factor (PDGF), which promote vascular smooth muscle cell proliferation and extracellular matrix deposition. The accumulation of smooth muscle cells and a proteoglycan-rich extracellular matrix in the intima beneath the endothelium is a process referred to as intimal thickening or intimal hyperplasia ( Figure 12.1 ). In humans, fatty streaks often form at sites where the intima is thus thickened. Within these lesions, the negatively charged proteoglycans are thought to bind the positively charged LDL particles, enhancing the retention of LDL within the intima and facilitating the phagocytosis of the LDL by macrophages.

Inflammation in Atherosclerosis

Inflammatory responses play important roles in the development of atherosclerosis. In atherosclerosis there is abnormal activation of inflammatory cells, which are largely directed towards the lipid deposited in the vascular wall. In general, the greater the systemic inflammatory response, the more likely there will be vascular-associated inflammatory reactions. Thus, atherosclerosis is amplified by a host of chronic inflammatory conditions including periodontal disease, rheumatoid arthritis, and systemic lupus erythematosus. In fact, current measures to stratify atherosclerotic risk include quantitation of non-specific inflammatory markers in the blood, such as C-reactive protein (CRP). People with a higher baseline CRP level have an increased risk for developing clinically significant atherosclerosis.

In atherosclerosis, the leukocytes in the circulation are characteristically recruited into the vascular wall at the sites of lipid accumulation. Initially the leukocytes are primarily monocytes, which become activated to form tissue macrophages. This activation is stimulated by cytokines secreted from the resident vascular cells and is also driven by the accumulated lipids. Modified LDL, for example, binds to macrophage scavenger receptors, becomes internalized, and then accumulates within these cells due to an inability to be appropriately digested by lysosomal enzymes. This accumulation results in the foam cell, a macrophage swollen with lipid vacuoles. Foam cells have abnormal activity, and cannot readily egress the vascular intima or migrate to lymph nodes. Indeed, lipid loading of these macrophages can trigger cell death that contributes to the accumulation of thrombogenic material within the intima, including phospholipid and tissue factor.

As an abortive attempt to remove or sequester abnormal lipid (perceived as a ‘danger signal’), foam cells recruit additional inflammatory cells, including pro-inflammatory cells such as T-helper type 1 (Th1) lymphocytes. Th1 lymphocytes secrete cytokines such as interferon-γ (IFNγ) and interleukin 2 (IL-2) that stimulate monocytes to form M1 polarized inflammatory macrophages. In turn, these inflammatory macrophages secrete large amounts of reactive oxygen species and catabolic extracellular enzymes including matrix metalloproteinases (MMPs) and myeloperoxidase (MPO). Although the inflammatory process can be counter-balanced by regulatory T lymphocytes (T-regs), and in some cases antibody-producing B lymphocytes, leukocytes continue to accumulate in the generally pro-inflammatory milieu. Consequently, more foam cells form, and more cytokines, proteases, and reactive oxygen species are expressed. Such cytokine expression drives smooth muscle cell and fibroblast proliferation, and also promotes endothelial activation, creating a positive feedback loop. In addition, MMPs can degrade the extracellular matrix in the vessel wall, promoting further vessel dysfunction, with exacerbation of the smooth muscle cell and fibroblast proliferation. Subsequently, the reactive oxygen and nitrogen species generated by inflammatory cells oxidize the entrapped LDL, further driving macrophage uptake via scavenger receptors.

The continued lipid accumulation, endothelial activation, inflammation, and vascular smooth muscle cell proliferation leads to a positive feedback loop that drives the growth of the atherosclerotic plaque. In some cases, this process eventually may stabilize, while in other cases it can accelerate. The particular balance of these processes at different anatomic sites will determine the exact clinical sequelae.

The Atherosclerotic Plaque

As lipid accumulates in foam cells, macrophage-derived cytokines, such as tumor necrosis factor α (TNF-α), further promote the recruitment and proliferation of smooth muscle cells and fibroblasts. In turn, these cells secrete large amounts of extracellular matrix including collagen and proteoglycans. A key step in the transition from a fatty streak to a more advanced atherosclerotic plaque is the accumulation of extracellular lipid ( Figure 12.1 ). Initially, this extracellular lipid interdigitates between the intimal smooth muscle cells. As the lesion progresses, the extracellular lipid coalesces to form large pools becoming the core of the atheroma. The core also contains necrotic material from dead foam cells and macrophages and is, thus, often referred to as the necrotic lipid core. Some of the lipid in the core originates from dead macrophage foam cells, while some entrapped LDL is likely deposited directly into the core. Within the lipid core cholesterol will crystallize to form sharp needle-like structures referred to as cholesterol clefts ( Figure 12.3 ). Atherosclerotic plaques frequently show evidence of hemorrhage. The blood often comes from small vessels that have grown into the plaque from the adventitia. The cholesterol in red blood cell membranes can add substantially to the cholesterol pool in the necrotic lipid core.

Fibroblasts are recruited into the plaque, possibly from the adventitia or from circulating precursors. These cells secrete large amounts of collagen, causing fibrosis or scarring. As the plaque matures, the combined necrotic lipid core and the surrounding scar matrix form the characteristic fibroatheroma. The lipid and collagenous content in atherosclerotic plaques is quite variable, even within the same patient. Some plaques are predominantly deformable necrotic cores covered by a thin fibrous cap (see discussion below), while others are essentially all fibrous tissue, referred to as fibrous plaques. As we will see, the actual architecture of plaques materially contribute to the subsequent pathologic outcomes. Advanced atherosclerotic plaques also undergo varying degrees of calcification.

Atherosclerotic plaques usually form with an eccentric geometry, that is, they are not uniformly distributed around the vascular circumference, but rather preferentially involve one portion of the vessel’s luminal circumference. This is explicable based on the eccentric nature of the disturbed shear stresses being imparted on the endothelium at branch sites. Initially there is often outward or positive remodeling of the arterial wall due to changes in smooth muscle cell numbers, vessel tone, and extracellular matrix deposition (Glagov phenomenon); this maintains the size of the lumen despite the encroachment of the atherosclerotic plaque. Correspondingly, atherosclerotic vessels exhibit cellular activation markers in the medial smooth muscle cells. However, there are limits to the adaptability of the vascular remodeling. When lesions occlude approximately 40% of the overall vessel diameter, the growth of the plaque begins to exceed the capacity to dilate, and there will be a diminished luminal cross-sectional area, so-called negative remodeling. At this point, such stenoses can be readily visualized by imaging techniques such as angiography and intravascular ultrasound.

The decreased luminal size restricts blood flow to the downstream tissue, although in most cases this will not become functionally limiting until 75% of the lumen is compromised. At this point, distal perfusion may be inadequate to supply the demands of the perfused tissue, leading to clinical symptoms, particularly when there is co-existent hypoxia, anemia, or hypotension.

Compromise of vascular flow in different sites becomes clinically apparent as distinct signs and symptoms. These include:

• Angina pectoris: insufficient blood flow to cardiac tissue classically results in chest pain; because of the vagaries of referred visceral pain (the pain sensation originates predominantly from pericardial innervation), angina may also be reflected by discomfort in the left arm or jaw. The pathogenesis of the pain is attributed to adenosine released from ischemic myocardium.

• Transient ischemic attacks (TIAs): insufficient blood flow to the brain can result in a host of neurologic sequelae including weakness, abnormal sensations, dysarthria (inability to speak), or syncope (loss of consciousness). If the insult is sufficiently mild or brief, there is full recovery. More significant compromise of perfusion leads to cerebrovascular accidents (stroke).

• Peripheral claudication: when blood flow to an extremity is compromised, there may be adequate perfusion at rest. However, any exertion (even simple ambulation) may lead to a supply–demand mismatch; with accumulating lactic acid (due to anaerobic metabolism), the muscles become painful.

• Increased blood pressure: diminished renal perfusion of the kidneys is perceived as a signal that there must be systemic hypotension. In such an instance, normal neuroendocrine feedback loops involving, for example, kidney renin release will lead to systemic hypertension by increasing vascular smooth muscle tone.

In some cases, however, the first symptoms relating to an atherosclerotic plaque may be catastrophic. General concepts are starting to emerge regarding the biological features of atherosclerosis that distinguish the gradual insidious forms of the disease from the sudden catastrophic manifestations. The current models distinguish between the stable atherosclerotic plaque as the source of slow progressive disease, and the unstable or vulnerable atherosclerotic plaque as the cause of sudden and catastrophic outcomes.

Unstable Plaques and Plaque Disruption

An unstable plaque is a plaque that undergoes a disruption of the endothelial surface causing a thrombus to form within the lumen of the vessel. This thrombus can rapidly narrow the lumen of the artery causing sudden catastrophic events, such as stroke or sudden cardiac death (the latter most commonly due to ischemia-induced arrhythmias). Such unstable plaques are also referred to as vulnerable plaques, because they are prone to disruption and thrombosis. Plaque disruptions can occur where there is previous severe stenosis of the lumen, with loss of around 75% or more of the cross-sectional area. Distressingly, such plaque disruptions can also occur where there is less than 70% chronic occlusion, and therefore when there is unlikely to be previous symptomatic ischemia. The resulting thrombus may entirely occlude the artery or only partially occlude the artery. In either case, fragments of the thrombus can embolize into the target tissue, obstructing smaller vessels and causing tissue ischemia.

Some of these plaque disruptions are due to full-thickness breaks, or ruptures, of the fibrous cap that overlies the necrotic and thrombogenic lipid core. In some fibroatheromas, the fibrous cap overlying the necrotic lipid core becomes markedly thinner due to the actions of matrix metalloproteinases. Such a plaque is often referred to as a thin cap fibroatheroma (TCFA) ( Figure 12.4 ). Full-thickness ruptures of the fibrous cap usually occur at the site of a TCFA ( Figures 12.5, 12.6 ). Upon rupture of the fibrous cap, the prothrombotic contents of the necrotic lipid core come into contact with the blood, triggering the clotting cascade and platelet aggregation. The ensuing thrombus markedly narrows the size of the previous lumen. Plaque ruptures are the most common form of plaque disruption, and since plaque ruptures most commonly occur in TCFAs, the TCFA is often considered to be the quintessential vulnerable plaque.

However, in addition to TCFAs, other forms of atherosclerotic plaque can also undergo acute plaque disruption. This typically occurs without a full-thickness break of the fibrous cap, and in these cases the process is termed plaque erosion ( Figure 12.7 ). In plaque erosions, the endothelial surface becomes disrupted and the underlying connective tissue triggers the formation of a thrombus. Unlike what happens in plaque ruptures, the entire necrotic lipid core does not come into contact with the blood. Thus, there is a tendency for the thrombus to be smaller in plaque erosions than it is in plaque ruptures. However, a plaque erosion can still cause significant acute narrowing of the vascular lumen as well as sudden catastrophic clinical events.

There is much interest in trying to understand the mechanisms triggering plaque rupture and erosion. Certainly in some patients, plaque disruptions appear to be related to an unusually exuberant inflammatory response in the atherosclerotic plaque ( Figures 12.8, 12.9 ). In essence, what were smoldering embers may in some patients flame up into a firestorm. Exactly why and how this occurs is not known. The inflammatory response in these situations involves increased infiltration of the atherosclerotic plaque by macrophages, dendritic cells, and even neutrophils. The inflammatory cells produce large amounts of MMPs that weaken the subendothelial connective tissue, in some cases causing rupture of the fibrous cap. In addition, the cytokines released by the inflammatory cells enhance endothelial dysfunction, promoting thrombosis. Many of the infiltrating inflammatory cells, both neutrophils and some macrophages, secrete myeloperoxidase in an attempt to destroy a perceived foreign invader. These observations are spurring efforts to identify circulating inflammatory markers in the blood, such as myeloperoxidase, which may be useful in identifying which patients are undergoing an exuberant inflammatory reaction in their atherosclerotic plaques.

While inflammation is clearly important in some cases of plaque disruption, sudden coronary artery thrombosis does occur in other patients by disruption of atherosclerotic plaques that contain few inflammatory cells. Thus, in these cases, alternative mechanisms of plaque disruption may be at work. Possible causes of plaque disruption in the absence of significant inflammation include vasospasm, endothelial degeneration, intraplaque hemorrhage, and physical protrusion of cholesterol crystals through the vessel wall elements. All of these mechanisms could disrupt the endothelial surface and promote the formation of luminal thrombus.