The Pathogenesis of Atherosclerosis and Plaque Instability

James S. Forrester

Despite the dramatic reduction in cardiac events reported in the lipid lowering trial, a substantial body of evidence from sources as diverse as epidemiology, clinical trials and cell biology suggests that the atherogenesis involves processes far more complex than elevation in serum lipids (Table 1.1). Until the 1980s the central focus of pathologists was the debate over whether coronary thrombosis is a premortem or postmortem event. In the late 1980s, however, coronary angioscopy in symptomatic patients focused attention on plaque rupture. Angioscopy in patients at the time they were experiencing clinical syndromes definitively demonstrated that the culprit lesion in patients with stable angina was an atheroma with a smooth surface, whereas those with unstable angina had a disrupted endothelial surface, with or without thrombus formation.1,2 Although these data established the causal importance of intimal disruption in acute coronary syndromes, there was no understanding of its pathologic basis.

In the early 1990s, vascular pathologists identified three characteristic histologic features of unstable plaque: a large lipid core, an abundance of inflammatory cells, and a thin fibrous cap.3 The differences in both size of the lipid core and macrophage volume between stable and disrupted plaques are striking. For instance, Felton et al. studied 334 human aortic plaques. In the aortae with disrupted plaque, the unstable lesions had fourfold greater cross-sectional area occupied by lipid, an eightfold greater area occupied by macrophages, and a fibrous less than a third as thick as that found in stable atheroma.4 Nonetheless, there was very limited insight into what biologic processes were responsible for the development of these three characteristics. At the turn of the century, therefore, there emerged a clear need to identify the cellular biologic processes which lead to the three unique histo-logic features of the unstable plaque.

In this chapter, we describe our current understanding of the cellular processes responsible for creation of the atheroma and its evolution to instability and rupture. These processes can be described didactically as a series of discrete steps (Fig. 1.1). This schema simplifies a complex process because a diverse group of mediators drive each step and each of the cell mediators affect more than one step in the plaque destabilization.

J.L. Holtzman (ed.), Atherosclerosis and Oxidant Stress: A New Perspective.

© Springer 2007

Table 1.1 Inferential evidence from diverse sources, which suggest that the lipid hypothesis is insufficient as a theory of atherogenesis

Inferential evidence

Major variation in death rates at same serum cholesterol level

Predicted vs observed trial outcomes

Substantial reduction in cardiac events with a diet that does not lower LDL

Reduced cardiac events with triglyceride lowering, with no change in LDL


Fourfold difference in cardiac mortality among countries in the same quartile of serum cholesterol53 35% greater reduction in events with statin therapy than predicted from an epidemiologic model54 30-70% short-term reduction in events post infarction with diets that have little or no effect on LDL 55 22% reduction in events in the VA HIT trial using gemfibrozil56

2. LDL & cells enter 1. Risk factors injure EC

  1. 1.1 The steps in atheroma destabilization. Activated endothelial cells express adhesion molecules that attract leukocytes that enter the blood vessel wall. LDL in the vessel wall is oxidized, and taken up by macrophages. The activated cells in the vessel wall express cytokines that maintain the inflammatory process. Proteases digest the fibrous cap, and smooth muscle cells undergo apoptosis, leading to rupture of the fibrous cap (see text)
  2. 1.1 The steps in atheroma destabilization. Activated endothelial cells express adhesion molecules that attract leukocytes that enter the blood vessel wall. LDL in the vessel wall is oxidized, and taken up by macrophages. The activated cells in the vessel wall express cytokines that maintain the inflammatory process. Proteases digest the fibrous cap, and smooth muscle cells undergo apoptosis, leading to rupture of the fibrous cap (see text)

Creation of the Lipid Core

The starting point for atheroma formation and plaque destabilization is endothelial activation. (In this chapter we use the term "activation" rather than "dysfunction," since cells frequently are responding normally to a noxious stimulus.) The activators of endothelial cells are the traditional risk factors for coronary heart disease (CHD) including hypercholesterolemia, smoking, and hypertension. But many other less well-recognized factors such as homocystinemia, immune complexes, and a wide spectrum of infectious agents also are capable of activating the endothelium.5,6 The response of the endothelium to these stresses is quite rapid. For instance, forearm vascular reactivity increases substantially in the 4-h period following ingestion of a fatty meal.7 Conversely, chronic low density lipoprotein (LDL) lowering improves vascular reactivity,8 and acute LDL apheresis can increase stress-induced coronary blood flow by 30% within 24 h of the procedure.

Endothelial cell activation is characterized by upregulation of leukocyte adhesion molecules and selectin adhesion receptors. This response may be particularly prominent at branching points of blood vessels, where the loss of normal laminar flow reduces the local expression of endothelium-derived nitric oxide, which suppresses adhesion molecule expression.810 In response, circulating white cells adhere and roll along the endothelial surface. After attachment, the cells express pseudopods and enter the blood vessel wall through the endothelial gap junctions. This movement is facilitated by monocyte chemoattractant protein-1 and other chemoattractants.11

The importance of this initial step at the blood cell-blood vessel interface in the initiation of atheroma formation is illustrated by studies in atherosclerosis-prone transgenic mice: animals deficient in platelet and endothelial selectins have 40% smaller lesions.12 In man adhesion molecule and selectin expression on plaques is twofold greater than on the normal arterial endothelium,13 and serum adhesion molecule concentration correlates directly with carotid intimal thickness as measured by ultrasound.14

Cholesterol moves in and out of the blood vessel wall attached to transport proteins. It enters as low density lipoprotein, with apolipoprotein B as its carrier. In the presence of local inflammation, the LDL that enters the blood vessel wall undergoes oxidation by oxygen-free radicals. Although the data from cell culture studies and animal models suggest that oxidation plays a central role in atherogenesis and plaque instability, the data in man is more inferential. Antibodies against LDL are found in atherosclerotic lesions, and human plasma contains antibodies that react with oxidized LDL.15 Further, Hasegawa et al. found that the level of plasma oxidized LDL increases with increasing age and is significantly higher in patients with atherosclerosis than controls.16 The process begins with peroxidation of polyunsaturated fatty acids in the LDL lipid.1718 These modified lipids are no longer recognized by the LDL receptor, but are recognized by the scavenger receptor of the monocytes that have entered the blood vessel wall. This receptor is not under sterol-mediated feedback control. Consequently, the monocytes avidly ingest cholesterol, and in the process become tissue macrophages. The differentiation from monocyte to macrophage is augmented by macrophage colony stimulating factor. Filled with lipid, these cells, appropriately named foam cells, become trapped as tissue macrophages in the sub-endothelial proteoglycan substrate of the extracellular matrix. Over time, the predominant lipids in the evolving atheroma become free cholesterol and cholesterol esters.

Lipid accumulation in the vessel wall is neither unidirectional nor relentless. It is balanced by reverse cholesterol transport, i.e., movement of cholesterol out of the blood vessel wall. This process also involves transport proteins and lipoprotein carriers. Reverse cholesterol transport begins with efflux of cholesterol from cell membranes to phospholipid acceptor particles in the interstitial fluid, the most important of which are nascent HDL particles, which are composed of phospholipid apo A-I. Cholesterol in the nascent HDL is esterified by lecithin cholesterol acyl-transferase (LCAT) to cholesterol esters. Cholesteryl ester transfer protein (CETP) exchanges the cholesteryl ester for the triglyceride, decreasing HDL-C. Cholesterol is then transported to the liver where it is excreted into the bile.

As a generality, as serum LDL increases, there is a compensatory increase in reverse cholesterol transport. For instance, de la Llera Moya found that patients in the highest decile of plasma LDL had a 30% greater rate of reverse cholesterol transport than those in the lowest decile.19 On the other hand, plasma HDL concentration correlates only roughly with the level of reverse cholesterol transport. Thus, because HDL also inhibits adhesion molecule expression, is an antioxidant and blocks matrix metalloproteinase expression, it has a number of potentially antiatherogenic actions.20,21

When lipid accumulation exceeds reverse cholesterol transport, the lipid core enlarges, creating the first histologic characteristic of the unstable plaque, the large lipid core. In compensation the external diameter of the vessel wall increases. This phenomenon has major clinical importance: serial angiographic studies before and after plaque rupture in man have that about half of the vulnerable plaques with large lipid cores are not flow limiting prior to plaque rupture. Thus unstable lesions are not necessarily severely stenotic, and conversely angiographically severe stenoses are not necessarily unstable.22

Local Inflammation in the Vessel Wall

Tissue macrophages, activated by oxidized LDL and/or other pro-oxidant stimuli, initiate and maintain a local inflammatory reaction by expression of cytokines.23,24 In the wall of the vessel with an unstable plaque, every cell type is activated (Table 1.2). The endothelial cell expresses adhesion molecules. Degranulating mast cells increase 15-fold and become TNF-alpha positive, serving as a potent stimulus to continuing endothelial cell activation.25,26 The smooth muscle cell changes from the contractile to the secretory phenotype, expressing extracellular matrix proteins, particularly collagen that forms the fibrous cap.27 This stabilizing effect, however, is countered by activated T-lymphocytes that express gamma interferon inhibiting extracellular matrix expression.28 In summary, the complete spectrum of inflammatory cytokines has now been identified in unstable human plaque.29-32 These cytokines

Table 1.2 Cell activation in the unstable plaque

Cell type

Unstable vs. Stable atheroma

Smooth Muscle

Two-fold increase in volume of synthetic organelles (42% vs. 21%)27


Eightfold greater volume of cells4


17:1 ratio of degranulated to granulated cells25


Along with macrophage, the predominant cell at rupture site29

There are major histologic and functional differences between stable and unstable human atheroma, even within the same vessel.

There are major histologic and functional differences between stable and unstable human atheroma, even within the same vessel.

have multiple overlapping actions. For instance TNF-alpha also promotes oxidative stress, TGF-beta stimulates the production of lipoprotein-trapping proteoglycans, colony stimulating factors cause macrophage replication, and interferon gamma suppresses smooth muscle replication.33 Cytokines that promote cap formation and stabilization, like platelet-derived growth factor and insulin-like growth factor, are also expressed,34 but in the unstable plaque the balance between these competing factors favors collagen breakdown rather than synthesis.

Reflecting the abundance and diversity of inflammatory cytokines, the temperature of unstable lesions is increased. For instance, in unstable angina patients the culprit lesion is on average 0.6°C higher than in patients with stable angina, and in patients with myocardial infarction it is 1.0°C higher.35 There is a direct correlation between plaque temperature and macrophage volume. In summary, the unstable plaque is the body's inflammatory process as it is expressed in the unique tissue of the blood vascular wall.

The process of plaque destabilization, however, is more complex than local inflammation alone. Systemic inflammation also plays an important, albeit less clearly defined role. Remarkably, local plaque temperature also correlates with the systemic level of circulating cell adhesion molecules, cytokines, and plasma c-reactive protein (CRP).36 Further, the presence of chromic infection and elevated CRP increases the risk of new atheroma formation fivefold.37 Chronic infection increases the risk of mortality in patients with established CHD by about 40%. A meta-analysis of seven studies involving 1,053 cases of non-fatal myocardial infarction or CHD death, with a mean follow-up of 6 years. The risk ratio of CHD for people in the upper tertile of plasma CRP compared to the bottom tertile was 1.7.38 Thus a reasonable speculation is that systemic inflammation aggravates local inflammation.

Thinning of the Fibrous Cap

The balance between connective tissue synthesis and breakdown determines the integrity of the fibrous cap that isolates the lipid core. The strength of the cap reflects extracellular matrix proteins expressed by smooth muscle cells. Opposing this action are the activated inflammatory cells, particularly macrophages, T-lymphocytes, and mast cells.39 Collagen digestion is accomplished by proteases, particularly the family of metalloproteinases (MMPs), expressed predominantly by macrophages.40 Indeed, in human unstable plaques macrophage density correlates with decreased mechanical strength.41 MMP expression is intimately related to LDL oxidation. In macrophages oxidized LDL doubles MMP expression whereas native LDL has no effect.42 Expression of MMP is also upregulated by tumor necrosis factoralpha and interleukin-1.43,44 The redundancy of the mechanisms responsible for plaque instability is illustrated by the spectrum of other compounds that induce MMP expression, including plasmin, oxygen radicals, and Chlamydial heat shock protein.45,46

Table 1.3 Destabilizing effects of cell products identified in unstable atheroma

Important action in unstable plaque

Other actions


Upregulates adhesion molecules

Increases thrombogenicity


Activates endothelial cells

Causes SMC apoptosis


Digests collagen

Digests elastin


Stimulates MMP expression

SMC apoptosis


Stimulates collagen synthesis

Stimulates lipid trapping proteoglycans


Promotes thrombin generation

Promotes MMP expression


Suppresses collagen expression

Causes SMC apoptosis

The cell products in unstable plaque each have multiple actions that contribute to stabilization. In addition, there is substantial overlap among the effects of cytokines. This redundancy makes it unlikely that targeting a single cytokine will be an effective approach. (Reproduced with permission from Forrester J. Ann Int Med 2002;137:823-833).

The cell products in unstable plaque each have multiple actions that contribute to stabilization. In addition, there is substantial overlap among the effects of cytokines. This redundancy makes it unlikely that targeting a single cytokine will be an effective approach. (Reproduced with permission from Forrester J. Ann Int Med 2002;137:823-833).

Concomitant with destruction of collagen in the unstable plaque, there is suppression of its synthesis. Smooth muscle cell function is suppressed by interferon-gamma from T-lymphocytes.47,48 The smooth muscle cells in advanced lesions also made susceptible to apoptosis by TNF-alpha and interferon-gamma. In our laboratory Wallner et al. have also shown that the extracellular protein tenascin-C, which is not present in the normal vessel wall, is strongly expressed by macrophages in unstable plaque.49,50 Tenascin stimulates MMP expression and causes smooth muscle cell apoptosis (Table 1.3).

Erosion of the fibrous cap culminates in plaque rupture, with release of tissue factor, followed by platelet adhesion and thrombus formation. As we observed by angioscopy a decade or more ago, if the thrombus is partially occlusive, it causes the syndrome of unstable angina, whereas complete occlusion causes myocardial infarction. Plaque rupture most commonly occurs at the plaque shoulder, where T-lymphocytes and macrophages predominate and smooth muscle cells are less common.51 Interestingly, tissue factor content in unstable plaque is twice that in stable plaques, and correlates directly with both macrophage volume,52 providing the final link the inflammatory process, plaque rupture and coronary thrombosis.

The pathogenesis of the three histologic characteristics of unstable plaque, so poorly understood just a decade ago, can now be defined. The formation of a large lipid core begins with LDL entry into the vessel wall. In the presence of chronic systemic or local vascular inflammation, created or amplified by a spectrum of risk factors, the endothelium is activated. Activated endothelial cells attract monocytes to enter the vessel wall. Within the vessel wall, the monocytes encounter oxidized LDL, the product of oxidative stress, also a manifestation of inflammatory activation. The monocytes avidly ingest oxidized LDL, becoming trapped in the suben-dothelium as tissue macrophages. As macrophages ingest LDL and later die, a large necrotic lipid core is created. The abundance of inflammatory cells in the unstable plaque is maintained and amplified by cytokine-induced cell activation. The third histologic characteristic of the unstable plaque is the thin fibrous cap. This results from extracellular matrix breakdown by proteases. At the same time collagen synthesis is diminished by cytokine-induced suppression of SMC function and promotion of SMC apoptosis. When the fibrous cap ruptures, most commonly at the shoulders, it exposes both tissue factor and collagen to the flowing blood stream. Both are prothrombotic.

In science, the tools we have for measuring it often determine the way we perceive reality. For CHD these perceptions have also determined management. In the 1980s, revascularization therapy had its origin in angiography. In the 1990s thrust of LDL lowering statin therapy had as its basis the correlation of elevated blood lipids and cardiac events. Today the ability to measure endothelial reactivity, oxidative stress, cholesterol transport, and serum and tissue cytokines provide the basis for an expanded view of management of CHD. Based on cell biology of plaque rupture, we can now identify at least five therapeutic targets for plaque stabilization: endothelial passivation, very aggressive LDL lowering, inhibition of LDL oxidation, acceleration of reverse cholesterol transport, and inhibition of inflammation. If these approaches are additive, substantial further reduction in coronary events should be possible in the coming decade.

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