7.1 The pathobiology of atherosclerosis

DINAH V. PARUMS

INTRODUCTION

Cardiovascular disease remains the chief cause of death in the United States and Western Europe, and atherosclerosis, the principal cause of myocardial and cerebral infarction, accounts for the majority of these deaths.

One of the problems in the study of the nature of human atherosclerosis lies in the lack of unanimity about the definition of the histopathological structure of the lesion. The name ‘atheroma’ was commonly used by the Greek writers to describe the yellow, intimal plaques or nodules containing ‘gruel-like’ material. ‘Arteriosclerosis’ was introduced in 1829 by Lobstein as a generic term for all diseases of the artery in which there is thickening of the vessel wall with induration  (Table 1) 163. Arteriosclerosis remains the acceptable collective term for what is known popularly as hardening of the arteries. The term ‘atherosclerosis’ was introduced by Marchand in 1904. The World Health Organization gives the definition of atherosclerosis as ‘a variable combination of changes of the intima of arteries consisting of focal accumulations of lipid, complex carbohydrates, blood and blood products, fibrous deposits and calcium deposits associated with medial changes.’

NORMAL ARTERIAL ANATOMY

Arteries are compliant, distensible structures with a flat internal surface. They consist of three layers; the intima, the media, and the adventitia.

At birth, the intima consists of a single layer of endothelial cells which rests on the basement membrane and is separated from the media by the internal elastic lamina. The media consists of interconnected smooth muscle cells which, in muscular arteries, are separated from the adventitia by the external elastic lamina. Large elastic arteries such as the aorta possess a media which contains numerous parallel elastin fibres. Vasa vasorum, found in the adventitia of larger arteries, provide oxygenation and nutrition to the outer layers of the artery.

Normal arterial physiology is shown in  Table 2 164.

NORMAL LIPID PHYSIOLOGY

Lipoproteins

Triglycerides and other lipids are insoluble in plasma and are therefore transported as lipoproteins, aggregates of variable size, lipid, and protein content. These lipoproteins are usually classified by their density on ultracentrifugation, the lipoprotein with the lowest density and the greatest triglyceride content having the highest flotation number  (Table 3) 165.

Apoproteins

Apoproteins are the lipid-free protein components of the plasma lipoproteins. They play a role in receptor recognition and enzyme regulation and maintain the structural integrity of the lipoprotein particles.

Apoproteins are divided into classes A, B, C, D, and E, and are further divided into subclasses  (Table 3) 165. Apoprotein A is the major protein in high-density lipoprotein. Apoprotein A1 binds phospholipid and activates lecithin cholesterol transferase.

Apoprotein B accounts for 90 per cent of the protein of low-density lipoproteins and is a major protein of chylomicrons and very-low-density lipoproteins. Apoprotein B has a role in the transport of triglycerides.

Apoprotein CII activates the lipoprotein lipase of adipose tissue, while apoprotein E is involved with recognition of the remnant particle by the liver.

The absorption of dietary fat

Dietary fat accounts for between 30 and 50 per cent of the energy intake of many people. In the small intestine, partial hydrolysis of fats occurs due to the action of lipases, and in the presence of bile salts, cholic and chenodeoxycholic acids, and some phospholipid, micelles are formed, followed by absorption of non-esterified fatty acids and monoglycerides in the duodenum and proximal jejunum.

Monoglycerides are re-esterified in the mucosal cells to form triglycerides. Dietary cholesterol esters are hydrolysed by pancreatic enzymes and cholesterol is absorbed in the small intestine where it combines with triglycerides, phospholipids, and specific apolipoproteins in the mucosal cells. The combination leads to the formation of triglyceride-rich chylomicrons which are secreted into the lymphatic circulation. Here, changes in cholesterol, phospholipid, and apoproteins occur, including the loss of apoprotein AII and uptake of apoproteins C and E.

Although triglycerides, phospholipids, and cholesterol have important functions in the body and are vital components of cell structure, raised levels of these lipids in the circulation are associated with an increased incidence of ischaemic heart disease.

Fat transport

Very-low-density lipoprotein is synthesized in the liver and is the form in which endogenously synthesized triglycerides are transported. Triglyceride is gradually removed from the chylomicrons and very-low-density lipoprotein by the action of lipoprotein lipase. This enzyme is present in adipose tissue and in capillaries at tall sites. Its activity is stimulated by apoprotein CII and also by insulin. Glycerides and non-esterified fatty acids which are released from chylomicrons are taken up by muscle, where they provide the main energy source for aerobic metabolism. Excess is stored as triglyceride in adipose tissue.

As triglycerides are removed, the remnant particle becomes smaller; some of the more water soluble components on the surface, such as phospholipid, unesterified cholesterol, and apoprotein C become redundant and transfer to high-density lipoprotein. The remaining chylomicron remnant is intermediate-density lipoprotein, some of which is metabolized by the liver and some of which is probably metabolized in the tissues to low-density lipoprotein.

Low-density lipoprotein is the main cholesterol carrier in the plasma, and is removed from the circulation at a much slower rate than that of many other particles. Low-density lipoprotein is bound to cells by high affinity receptors. When it enters the cell it is degraded in lysosomes to liberate cholesterol: dietary cholesterol inhibits the activity of enzymes responsible for endogenous cholesterol synthesis. The number of cell receptors is regulated by intracellular cholesterol levels.

High-density lipoproteins form the other group of lipoproteins in the circulation; these are mainly synthesized in the liver and intestinal mucosa. Phospholipids and cholesterol are transferred to high-density lipoproteins, where cholesterol esters are formed by the action of lecithin cholesterol acyl transferase.

THE FUNCTION OF LIPIDS

Triglycerides

These are derived from animal and plant dietary sources and account for up to 95 per cent of the lipids in adipose tissue. They are a source of energy during periods of starvation. During periods of adequate feeding, triglycerides can be synthesized in the body and stored.

Phospholipids

Phospholipids are fundamental components of cell membranes.

Cholesterol

Dietary cholesterol is derived mainly from dairy products and eggs: diets high in saturated fat are generally high in cholesterol. The average intake in people eating Western diets is 300 to 500 mg per day, but it may be as high as 1000 mg.

Cholesterol is also synthesized in the body and is excreted in bile salts and bile as free cholesterol.

Cholesterol is an important component of cell membranes and is particularly involved in the regulation of membrane fluidity and stability. It is transported round the body as a component of lipoproteins and is also an important precursor of steroid hormones and bile acids.

No other blood constituent varies so much between or within populations as plasma cholesterol level, with a range of 100 to 275 mg/dl (2.6–7.1 mmol/1).

HYPERLIPIDAEMIA

A high level of circulating lipoproteins usually results from an increase in their synthesis due to a diet high in saturated fat and/or a genetically determined reduction in their removal from the circulation. Depending on the type of particles this causes an increase in the concentration of cholesterol and/or triglycerides in the plasma.  Table 4 166 is one classification of familial hyperlipidaemia, based on the World Health Organization (Frederickson) classification. Familial hyperlipidaemia is one of the most common inherited conditions, affecting at least 1 in every 500 people in the United Kingdom. In some populations, such as Lebanese and Afrikaaners, the incidence is much higher. It is inherited in an autosomal dominant manner.

Conditions which may cause secondary hyperlipidaemia include diabetes mellitus, hypothyroidism, excessive alcohol intake, obesity, nephrotic syndrome, pregnancy, biliary obstruction, myeloma, and intake of drugs such as thiazide, steroids, &bgr;-blockers, and oral contraceptives.

EPIDEMIOLOGY OF ATHEROSCLEROSIS

Incidence

Atherosclerosis and its complications are the leading cause of morbidity and mortality in the Western world, accounting for more than 50 per cent of all deaths. Over 80 per cent of these deaths are due to arteriosclerosis and hypertension combined.

Prevalence

Atherosclerosis shows a prevalence of nearly 100 per cent in adults. The severity of the disease varies from mild to severe when comparisons are made between groups, individuals, and even within individuals. In general, atherosclerosis increases with age, but it is not thought to be an intrinsic biological ageing process as most mammalian species age without spontaneously developing atherosclerosis.

Males are affected more frequently than females, but the differences tend to diminish with increasing age: the ratio of affected males to females is 6:1 at ages 35 to 44, but 2:1 in the 65 to 74 age group.

Heredity

Heredity influences the severity of atherosclerosis directly by affecting arterial wall structure and function and indirectly through such factors as hypertension, hyperlipidaemia, diabetes, and obesity.

Risk factors

Epidemiological studies (such as the Framingham study) show that certain habits, diseases, and lifestyles are more important than others and offer different degrees of risk  (Table 5) 167. It must be realized that advanced atherosclerosis and its clinical complications are uniquely human conditions. It is not possible to follow the progression of atherosclerosis within an individual, and epidemiology has to rely on the assessment of clinical consequences of atherosclerosis, such as myocardial infarction, as they apply to populations. Although these risk factors may be important in the development of these clinical complications; they do not necessarily per se reflect what is going on at the level of the intimal lesion in a single individual.

PATHOLOGY

Types of lesions

The lesions seen in atherosclerosis consist of fatty streaks, fibrous plaques, and complicated or advanced plaques.

Fatty streaks are yellow, flat lesions arising between the intima and internal elastic lamina, consisting of macrophages containing cholesterol and cholesterol esters derived from plasma. Although they occur at all ages, fatty streaks are most commonly seen in the aorta of children. These lesions can regress, and there is still debate as to whether they progress to advanced plaques.

Fibrous plaques are grey-white, elevated lesions consisting of subendothelial proliferations of smooth muscle cells, collagen, and variable amounts of extracellular lipid  (Fig. 1) 200. They appear in the second and third decades of life at bifurcation points in arteries and the aorta.

Complicated/advanced plaques are pale yellow-grey or white raised lesions of varying size affecting the intima and inner media. They give rise to local complications  (Fig. 2) 201 and are clinically the most important type of lesion.

The local sequelae of the advanced plaque give rise to the clinical complications of atherosclerosis, most commonly ischaemia and infarction.

The local complications of advanced atherosclerosis include stenosis of the arterial or aortic lumen, plaque ulceration and fissuring (with or without atheroemboli), thrombosis (with or without thromboemboli), calcification, haemorrhage into the plaque, aneurysm formation (with or without thrombosis and thromboemboli), and chronic inflammation (chronic periaortitis) see  Section 7.2 34 ( Fig. 2 201.)

Patterns of lesion distribution

The abdominal aorta is affected more often than the thoracic aorta. Atherosclerosis is particularly seen around ostia of branch vessels. It is rare in pulmonary arteries, except in the presence of pulmonary hypertension.

Major anatomical patterns include involvement of coronary arteries, the terminal abdominal aorta and its branches, the innominate, carotid, and subclavian arteries and their branches, and visceral branches of the abdominal aorta including the renal arteries.

Although these patterns of distribution are fairly characteristic, clinical experience suggests that there is some selectivity in their occurrence in different categories of patients. Some patients, for example, are prone to cerebrovascular disease with little or no evidence of disease at other sites. It is unclear which factors are most important in determining anatomic patterns of involvement.

Pathogenesis of atherosclerosis

Multiple theories of atherogenesis have been proposed  (Table 6) 168. Perhaps the earliest and best known theories were those elaborated in 1844 by Carl von Rokitansky (the thrombogenic theory) and in 1835 by Rudolph Virchow (the lipid inhibition theory). Virchow also believed that atheroma was a chronic inflammatory process involving the intima. While it is now evident that platelets, fibrin, lipids and mononuclear cells do play a part in atherogenesis, the key question is, how? Many theories of atherogenesis still abound; it is likely that multiple factors which affect the status of the arterial wall and the composition and dynamics of the blood are involved.

In the past decade, the cellular nature of atherosclerosis has been realized and more clearly understood. Many immunological and molecular biology studies have been performed on experimentally induced lesions in animal models but an increasing amount of research is being undertaken in man. We are only just beginning to understand how hypercholesterolaemia and hypertension might lead to the development of atherosclerosis.

It is now clear that the principal changes that take place in the artery wall during atherogenesis occur largely within the intima of medium and large arteries. The key factors include the entry of cells and non-cellular substances, including lipids (principally low-density lipoproteins), from the plasma.

Role of the arterial wall

Intimal injury and smooth muscle cell proliferation

Injury to the intima causes proliferation of smooth muscle cells and myofibroblasts (cells with phenotypic characteristics of both smooth muscle cells and fibroblasts) within the intima. Proliferation can be seen in experimental animal models and in man as part of an age-related phenomenon, known as diffuse intimal thickening. Cultured smooth muscle cells in vitro are capable of synthesizing extracellular matrix components such as collagen, elastin, and mucopolysaccharides. Smooth muscle cells can also metabolize lipoproteins and accumulate cholesterol esters.

Reponse to injury theory

Mechanical, chemical, or immunological damage to the endothelium, results in entry of plasma constituents such as lipoproteins and fibrinogen, together with cellular elements including platelets, monocytes, and lymphocytes. Platelet-derived growth factor can induce smooth muscle cell proliferation, forming a plaque which then progresses due to lipid infiltration and modification, further proliferation of monocytes, platelets and lymphocytes, and smooth muscle cell proliferation and collagen production and degradation.

Monoclonal theory

Cells in atheromatous plaques of black females were observed by Benditt and coworkers to be monotypic for the A or B isoenzyme of glucose 6-phosphate dehydrogenase. This was interpreted as evidence that atherosclerosis represents a neoplastic intimal lesion. There is, however, a recognized tendency to monotypism in other benign, non-neoplastic proliferative lesions, such as scar tissue.

Viral theory

Herpes virus particles and viral DNA can be detected in early atherosclerotic lesions in humans. How this relates to atherogenesis is still unclear.

Role of lipids

Dietary evidence

This remains a surprisingly controversial area. Dietary fat intake is probably important, since human populations consuming typical diets high in saturated fats and cholesterol have high mean serum cholesterol levels and have high mortality rates from coronary artery disease.

Recent studies performed on large numbers of hypercholesterolaemic individuals who have been treated with drugs that reduce plasma cholesterol levels have shown that decreasing cholesterol levels over time decreases the incidence of the clinical sequelae of atherosclerosis. As a result, guidelines for the reduction of serum cholesterol levels have been drawn up. Plasma cholesterol levels below 200 mg/dl are acceptable; levels between 200 and 240 mg/dl should be treated by diet; levels above 240 mg/dl should be treated by diet together with cholesterol lowing agents such as 3-hydroxy–3-methylglutaryl coenzyme A reductase and bile acid sequestrants.

Hyperlipidaemia

Patients with genetically determined hyperlipoproteinaemia  (Table 4) 166 and marked hypercholesterolaemia due to nephrotic syndrome, diabetes mellitus, and untreated myxoedema, have severe atherosclerosis. However, the correlation between the severity of atherosclerosis and cholesterol levels within an individual is imperfect: the state of circulating lipids, rather than the level, may be of importance.

Low-density lipoprotein

Low-density lipoprotein is the main carrier of plasma cholesterol to the tissues of the body. Studies on patients with familial hypercholesterolaemia have shown that hepatocytes, fibroblasts, and smooth muscle cells carry high affinity receptors for plasma low-density lipoprotein, which are down-regulated when this lipoprotein is plentiful. Patients with familial hypercholesterolaemia have defective receptors.

Low-density lipoprotein can be modified by malonation and oxidation, principally by macrophages or exogenous agents. Macrophages, endothelial cells, and smooth muscle cells also contain non-saturable, ‘scavenger receptors’ or ‘modified low-density lipoprotein receptors’ which are not down-regulated and which preferentially take up modified low-density lipoprotein.

High levels of circulating high-density lipoprotein appear to be protective, even in individuals with raised cholesterol levels.

Oxidized lipids

Ceroid is the name given to the insoluble yellowish pigment present in mammalian tissues, especially in the presence of vitamin E deficiency. It is regularly seen in association with human atherosclerotic plaques and can be regarded as the hallmark of the advanced lesion. It is insoluble in lipid solvents and is therefore recognizable in routinely processed tissue sections by lipid stains such as Oil Red O  (Fig. 3) 202. It is thought to consist of polymerized products of oxidized lipoproteins, predominantly low-density lipoproteins, within macrophages.

Oxidized lipids are toxic and immunogenic: they can act as chemoattractants for leucocytes and induce cell proliferation. Their effects could account for the progression and some of the complications of atherosclerosis.

Experimental/animal models

Lesions resembling atherosclerosis can be induced in experimental animals by a combination of high lipid diets and intimal injury.

Role of cells in atherosclerosis

Endothelial cells

The endothelium is able to modify and transport lipoproteins, to form vasoactive substances, to participate in leucocyte adherence, to produce growth factors, and to participate in procoagulant and anticoagulant activity.

Injury to endothelial cells or exposure to cytokines can induce endothelial cells to express genes for mitogens such as platelet-derived growth factor and interleukin-1. This may be of importance in the progression from early to advanced atherosclerotic plaques. Endothelial cells express class II major histocompatibility antigens and are involved in antigen presentation; this may be of importance in immunologically induced endothelial damage and in recruitment of lymphocytes into the lesion.

In advanced plaques, the endothelium no longer remains, but new vessel formation is seen at the base of the plaque. The endothelial cells at these sites may perform similar functions.

Smooth muscle cells

Smooth muscle cells are the principal source of collagen in the fibrous plaque; they can take up and modify lipoprotein and they are an important source of platelet-derived growth factor. Smooth muscle cells are present in diffuse intimal thickening and their numbers are increase in larger lesions.

Macrophages/monocytes—the macrophage hypothesis

Foam cells, which are present in fatty streaks and at the edges of most advanced plaques, are macrophages  (Fig. 4) 203 and macrophages are found in the necrotic base of the advanced atherosclerotic plaque.

In terms of the development of the clinical complications of atherosclerosis, the most important roles of the macrophage include their interactions with lipoproteins—secretion of monokines which recruit and modulate the behaviour of other cells (platelet-derived growth factor, fibroblast growth factor, transforming growth factor &agr; and &bgr;, colony-stimulating factor-1, tumour necrosis factor, and interleukin-1), release of enzymes; release of oxygen radicals, and their ability to modify lipoprotein, rendering it toxic, immunogenic, and more amenable to the scavenger receptor pathway.

Platelets

Platelets produce growth factors and mitogens, the best known being platelet-derived growth factor, an endothelium-derived growth factor-like substance, and transforming growth factor-&bgr;. Platelets play an important role in thrombosis and in the coagulation process: they may be sources of mitogens during the development of early atherosclerotic lesions, when mural thrombus forms at sites of endothelial damage. In advanced plaques, lesions may progress at sites of fissuring due to secondary thrombosis, which may, in turn lead to smooth muscle cell proliferation and collagen deposition.

Lymphocytes

T lymphocytes, but not B lymphocytes, are present within early and advanced atherosclerotic plaques, but the reason for their presence remains unclear: it may imply that immunological phenomena are involved in atherogenesis or in the progression of atherosclerosis. These lymphocytes are activated and strongly express major histocompatibility class II antigens.

Both T and B lymphocytes are seen in the adventitia in chronic periaortitis  (see Section 7.2) 34, when the atheroma thins the media.

Role of haemodynamic factors

The location of fibrous plaques at bifurcation points and branch points can be best explained by consideration of increased haemodynamic forces at these sites. There is a direct relation between hypertension and atherosclerosis in systemic arteries. Atherosclerosis is only seen in the pulmonary arteries in association with pulmonary hypertension.

Role of thrombogenic factors

Mural thrombi may become organized to the endothelium and resemble fibrous plaques. Fibrin and platelets are associated with developing plaques, and thrombosis may be important in their extension. Intraplaque haemorrhage and fissuring exposes collagen, which is highly thrombogenic.

Do theories of atherogenesis explain clinical risk factors?

Hyperlipidaemia

Low-density lipoprotein is the source of lipid in early atherosclerotic plaques and of the large amounts of cholesterol in advanced plaques. Individuals with Type II or Type IV hyperlipoproteinaemia  (Table 4) 166 have more atherosclerosis. These individuals have high circulating levels of low-density lipoprotein without necessarily having a raised serum cholesterol.

Cigarette smoking

Cigarette smoking is the factor with the strongest epidemiological association with the incidence and severity of atherosclerosis. A series of glycoproteins derived from tobacco has been associated with an immune response within the vessel wall. Increased serum concentrations of carbon monoxide in smokers are also thought to be injurious to the endothelium.

Hypertension

Hypertension acts synergistically with other risk factors for atherosclerosis. Altered haemodynamic properties of blood flow, causing endothelial injury, and humoral mediators of blood pressure, such as renin and angiotensin may be involved. The specific mechanisms by which hypertension increases the severity of atherosclerosis, however, still remain unclear.

Diabetes mellitus

Many diabetic individuals are hypercholesterolaemic. The mechanisms underlying the increased severity of atherosclerosis seen in those who have normal cholesterol levels are unknown. Some diabetics have decreased levels of high-density lipoprotein and are often hypertensive. Specific factors in the arterial wall or present in the plasma of diabetics may account for these observations remain unidentified.

Regression of atherosclerosis

Although fatty streaks experimentally induced in animals are reversible, there is still controversy over whether advanced atherosclerotic lesions in humans can regress. Some studies have shown that angiographically demonstrable lesions in hypercholesterolaemic patients, become smaller with a combination of diet and cholesterol-lowering drugs.

A possible mechanism by which regression may occur is by lipid-laden intimal macrophages re-emerging into the blood, although there is no evidence that this occurs in man.

FURTHER READING

Ball M, Mann J. Lipids and Heart Disease: a Practical Approach. Oxford: University Press, 1988.

Benditt EP, Benditt JM. Evidence for a monoclonal origin of human atherosclerotic plaques. Proc Natl Acad Sci USA, 1973; 70: 1753–56.

Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science, 1986; 232: 34–47.

Mitchinson MJ, Ball RY. Macrophages and atherogenesis. Lancet, 1987; ii: 146–9.