The

Lipid Hypothesis Pathophysiological Jean

\s=b\ A detailed

analysis

of the multifactorial

Basis

Davignon, MD, MSc, FRCP(C)

pathogenesis

of

atherosclerosis, including endothelial injury, proliferation of intimal smooth muscle cells, the role of the platelet, the role of cholesterol as raw material and irritant, the influence of inflammation, and the participation of lysosomal components, is presented. (Arch Surg 113:28-34, 1978)

multifactorial pathogenesis of atherosclerosis is well recognized.1 In the complex chain of events that leads to the development of an atheromatous plaque, three levels of influence may be considered (Fig 1). First, the ecological and genetic environment provides a favor¬ able setting through dietary constituents, risk factors, and some acquired or inherited diseases. At a second level of influence, these factors interact with the arterial wall through some components of the circulating blood to promote the formation of a plaque. Finally, constituents of the arterial wall take part in the process, either in combating or exacerbating the atherogenic influence. Dietary saturated fats, cholesterol, sucrose, animal

The

now

proteins or excess calories, cigarette smoking, hyperten¬ sion, hyperlipidemia, and diabetes are but a few of the environmental factors that may presumably be involved. Their effect on the arterial wall may be mediated through, for example, low-density lipoproteins, lowered oxygen tension, rheological factors, insulin, or platelets. On the other hand, the response of the artery will depend on the permeability of the endothelium, the structure of the arterial wall, and its biochemical and metabolic activity. Ever since Rudolph Virchow proposed the insudation theory in the 19th century, a large body of evidence has accumulated to give cholesterol a central role in this chain of events. In recent years, some aspects of the lipid hypothesis have been challenged, but valuable new knowl¬ edge has been gained on the different roles of the choles¬ terol-bearing low-density and high-density lipoproteins. Investigators have become more and more concerned with the initiating events in atherogenesis and the emphasis has been placed on the contribution of endothelial injury, the proliferation of intimai smooth-muscle cells, and Accepted for publication July 1, 1977. From the Department of Lipid Metabolism and Atherosclerosis Research,

Clinical Research Institute of Montreal, the Section of Vascular Medicine, H\l=o^\tel-DieuHospital of Montreal, and the Faculty of Medicine, University of Montreal. Reprint requests to 110 Pine Ave W, Montreal, Quebec, H2W 1R7, Canada.

function. This brief review summarizes some of aspects recent developments that have produced chal¬ lenging new hypotheses. For further details, the reader is referred to more comprehensive recent reviews.17

platelet

CENTRAL ROLE OF CHOLESTEROL importance given to plasma cholesterol and to its vehicle in man, the low-density lipoproteins (LDL), major stems from a large number of pathological, epidemiological, and experimental observations1-4: 1. Cholesterol is a major constituent of the plaque and is derived mainly from plasma LDL that accumulate in atheromatous lesions. 2. Manipulations that increase plasma cholesterol con¬ centrations can induce atherosclerotic lesions in animals. The resemblance of these lesions to human atherosclerosis has been questioned,8 but it is now known that they may be induced not only in herbivorous species, eg, the rabbit, but also in omnivorous animals and in primates. In the species that are closer to man, there is no need to resort to the induction of hypothyroidism and the feeding of large amounts of cholesterol. Indeed, atherosclerotic lesions similar to those found in man have been produced in rhesus monkeys fed diets resembling the usual American diet. 3. Many epidemiological studies have found a strong correlation between plasma cholesterol concentrations and the prevalence of coronary atherosclerosis. 4. Patients with familial hyperlipidemia are more likely candidates for the development of atherosclerosis and its complications. The lesions are usually more severe and develop earlier in life when the LDL-cholesterol level is above normal, as in familial hypercholesterolemia type II. 5. Measures aimed at lowering plasma cholesterol concentrations in experimental hypercholesterolemia and atherosclerosis have been shown to reduce the size of the lesions. There is more and more direct and indirect evidence to indicate that such a regression takes place in man with a number of cholesterol-lowering measures.910 The exact mechanism relating cholesterol to the athero¬ genic process is unknown. Cholesterol transported by LDL could be considered as a chemical agent capable, when its concentration is high enough in plasma and/or when other injurious elements weaken the endothelial barrier (hemodynamic stress, carbon monoxide, catecholamines, etc), of injuring the endothelium and infiltrating the intima to induce the proliferation of smooth-muscle cells.12 It is known that diets rich in cholesterol increase the permeaThe

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bility of aortic endothelium to proteins prior to the onset of

atherosclerosis in rabbits. Furthermore, the feeding of cholesterol for periods as brief as three days have been shown to increase the turnover of aortic endothelial cells in the swine as the plasma cholesterol level is rising. That local injury to the endothelium may be induced by circulating LDL-cholesterol is also borne out from a recent study of Ross and Harker.11 When they increased the plasma cholesterol concentration by feeding monkeys a diet high in cholesterol, about 7% of the intima of major arteries suffered damage, whereas the arteries of control monkeys remained intact. Low-density lipoproteins have been shown not only to promote the proliferation of human arterial smooth-muscle cells in culture, but also to be readily taken up by such cells. It is thus possible that a sustained endothelial injury from exposure of the arterial wall to chronically elevated concentrations of LDL-choles¬ terol could constitute an initiating factor in atherogenesis. Further accumulation of lipids in the proliferating intima could be accounted for by a defective removal secondary to binding of LDL to glucosaminoglycans, to an inadequate catabolism of the cholesteryl esters in the LDL, or to the formation of insoluble cholesterol crystals. This defective removal could be further hampered by the sclerogenous properties of cholesterol and of some of its esters.

Receptors and Remnant Lipoproteins Brown and Goldstein,12 working with human fibroblasts in culture, have shown that they contain a cell-surface receptor that binds and degrades plasma LDL. This has further emphasized the importance of LDL in atherogene¬ sis. These LDL receptors appear to regulate the cholesterol LDL

by modulating the rate of cholesterol uptake, esterification, and synthesis. After LDL becomes bound to the receptor of normal cells, the membrane invaginates to form a pinocytic vesicle that engulfs the LDL. These vesicles merge with the lysosomes, whose enzymes digest the protein moiety and hydrolyze the cholesteryl ester portion (mostly linoleate). The free choles¬ terol thus released is partly used for membrane formation; it also reduces the intracellular synthesis of cholesterol by suppressing hydroxylmethyl-glutaryl-coenzyme A reduc¬ íase and activates acylcoenzyme A cholesteryl acyltransferase, which catalyzes the reesterification of cholesterol for storage (mostly as oléate and palmitoleate). This cholesterol should also suppress the synthesis of LDL content of the cell

receptors, and thus prevent accumulation of

too much cholesterol by the cell. Patients with familial hypercholes¬ terolemia type II presumably have few of these receptors, and subjects homozygous for this disease would have

virtually none.

The LDL receptor has also been demonstrated in culture the surface of cells from human aortic media. Goldstein and Brown13 have postulated a role for LDL receptors in the development of atherosclerosis in normocholesterolemic subjects (Fig 2). According to their hypothesis, choles¬ teryl esters are formed slowly in the cells of the arterial wall, because the LDL receptors of these cells are not saturated by the low concentrations of LDL in the surrounding fluid. When the endothelial lining is damaged (by any mechanism), LDL particles pass more quickly into the interstitial fluid. The receptors then bind larger

on

LDL, which stimulate intracellular esterification of cholesterol. The consequent abnormal accumulation of cholesteryl esters within the intimai smooth-muscle cells would result in the formation of foam cells and plaques. While epidemiological data continue to support the concept that high LDL concentrations may contribute to the development of atherosclerosis and coronary heart disease, more and more evidence indicates that high concentrations of high-density lipoproteins (HDL, a-lipoproteins), in contrast, afford some protection.1'114 (This question is discussed in more detail in the next article of this symposium.) The role of elevated plasma triglycéride levels in atherogenesis is still a matter of debate.15 The recent hypothesis of Zilversmit18 that lipoprotein "remnants" derived from triglyceride-rich lipoproteins might play a part in the development of atherosclerosis is attractive. According to his view, chylomicrons and very-low-density lipoproteins (VLDL) are focally adsorbed at the endothelial surface of the arterial wall in proportion to the local concentration of sulfated polysaccharides. Lipoprotein lipase, present in situ, strips these molecules of their triglycérides and changes their lipids and protein composition as their size is reduced. The resulting "indigestible" remnants, which are rich in cholesteryl esters, may be partially released into the bloodstream and partially taken up by endothelial or intimai cells through a phagocytic or pinocytic mechanism. The uptake might be preferential for some degradation product of the lipase reaction, or it might be enhanced by factors capable of altering the endothelium. The lipolytic process would maintain a high concentration of cholesterolrich particles at the blood-artery interface and supply fatty-acid anions that would facilitate cholesterol or lipo¬ protein transport across the endothelial barrier. The excess cholesteryl ester thus taken up by the cell could overwhelm the ability of the lysosomal cholesteryl ester hydrolase to dispose of it and accumulate, with resultant foam cell formation and atherosclerosis. This "lipoprotein lipase mechanism" would reconcile the reported absence of exces¬ sive triglycérides from arterial lesions with a presumed involvement of hypertriglyceridemia in atherogenesis. It could also account for the development of atherosclerosis in the absence of fasting hyperlipidemia. amounts of

ENDOTHELIAL INJURY AND ARTERIAL WALL STRUCTURE

The endothelium offers a natural barrier to the infiltra¬ tion of unwanted substances from the blood and controls the passage of large molecules into the inner layers of the arterial wall. In the large- and medium-sized arteries, the avascular inner two thirds of the wall are highly dependent on the selective permeability of this continuous monolayer of cells for the delivery of oxygen and essential nutrients. Thus, any alteration of the endothelial lining may have important repercussions on the subjacent intima and inner medial layer, which are involved in the formation of atheromatous plaques. It is not surprising that many investigators of atherogenesis have looked at the endothe¬ lium as the essential site for the initiating event.1-8-' It has been hypothesized that plaques are formed in response to sustained or frequently recurring injuries to the inner lining of the artery. The "response-to-injury" hypothesis is

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DIABETES

DIET

CIGARETTE SMOKING

rCHOLESTEROL

SATURATED FATS VIRUS CHO PEANUT OIL VIRUS?

FOAM CELL FORMATION

ce

'^fecrtSTöP^r^AöWVASA VASÔRUM¿^lymphaticsTXry.r) Oxhin^yh°A^

Fig 1.—Multifactorial interactions in atherogenesis. Three levels of influence are depicted: ecological and genetic factors, intermediates in circulating blood, and arterial wall components. Endothelial injury and intimai cell proliferation are emphasized as key elements in atherogenic process. CHO indicates carbohydrates; Ab/ Ag, immune complexes; A-ll, angiotensin II; EPI, epinephrine; NE, norepinephrine; VLDL, very-low-density lipoproteins; LDL, low-density lipoproteins; TG, triglycérides; CE, cholesteryl esters; LPL, lipoprotein lipase; LCAT, lecithin/cholesterol acyltransferase; and GAG, glycosaminoglycans (from Davignon1).

supported by the fact that almost any kind of chronic damage to the artery, whether mechanical, immunological, or chemical, will induce in the experimental animal lesions

that resemble human atherosclerosis. Such lesions may develop not only from harshly injurious stimuli, such as stripping of the endothelium with a balloon catheter or the removal of endothelial patches with homocystine infusions, but also from subtler injuries. In recent years, new techniques have been developed to demonstrate minor alterations in endothelial permeability and obtain more information on the changes taking place early in the formation of a plaque. Vascular surgeons, as well as angioradiologists, are well aware of the sites of predilection for the development of atheromatous lesions. Hemodynamic factors such as increased turbulence, lateral pressure, and shearing stress have been held responsible for the preferential distribution of plaques in areas characterized by curvature, bifurcation, branching, tapering, and external attachments. In the past few years, the Evans blue dye technique has been applied to the study of hemodynamic factors and their early and late morphological and metabolic effects on the arterial wall.121718 This azo dye binds to serum albumin and

becomes

convenient visual tag for the study of albumin the endothelial surface or the accumula¬ tion of macromolecules in the intima at sites of endothelial injury and increased permeability. Administration of this dye in vivo produces in normal animals a focal spontaneous pattern of blue-stained areas that, in a given species, is virtually identical to the pattern of sudanophilia seen in early experimental atherosclerosis. This pattern of dye uptake, which cannot be reproduced by adding the albumin-bound dye in vitro to the dissected aorta, has been attributed to regional disturbances in blood flow. It may be altered by changing the geometry of the arterial tree. Thus, a pattern of dye uptake may be obtained in experimental aortic coarctation in the pig that closely mimics the pattern of development of atheroscle¬ rosis in aortic coarctation in man. In acute experiments, it has been shown that the flux of Evans blue dye into the intima is increased with increased stretch or wall strain, increased shearing stress, or increased turbulence. It appears that the flux of Evans blue dye can be increased in this form of injury before endothelial structural alterations become evident. Study of Evans blue dye uptake has focused attention on important regional differences in the a

transport

across

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DAMAGE TO

0

LDL n

PLASMA

Fig 2.—Low-density lipoprotein (LDL) receptors and atherogeneDamage to endothelium enhances permeability to LDL mole¬ cules, which pass into interstitial fluid at a higher rate. Receptors on intimacytes bind higher amounts of LDL, which stimulate intracellular fatty acylcoenzyme A (acyl-CoA)/cholesteryl acyltransferase (ACAT) activity, increasing cholesterol esterification. Consequent abnormal intracellular accumulation of cholesteryl esters (CE) would result in formation of foam cells and plaques.13 sis.

and metabolism of the arterial wall. Compar¬ ison of "blue" (stained) and "white" (unstained) areas has shown that more albumin and free cholesterol are taken up by the blue areas. They further exhibit an increased endothelial cell turnover and a greater uptake of fibrinogen. Cholesterol feeding of pigs for six weeks results in an accumulation of cholesterol in the blue, but not in the white, areas, which suggests that the two areas differ in their permeability to lipoprotein cholesterol. Furthermore, cholesteryl ester formation is significantly greater in the blue areas in the first few weeks of cholesterol feeding. These findings are in keeping with the concept of endothe¬ lial damage as an initiating event in atherogenesis. This concept is further supported by the finding that early injections of colloidal iron particles in lipid-fed animals are demonstrable in the newly developing lipid-filled lesions, but not elsewhere in the intact intima.

physiology

Mechanical

Injury

The response of the arterial wall to a mechanical injury depends on the type, force, and duration of the applied stress.181" A shear stress applied steadily for a long time in one direction will result in intimai fibrosis characterized by a dense, highly oriented subendothelial collagenous sheet, with few smooth-muscle and connective-tissue cells. These regions of repair become less permeable to protein and almost never contain stainable lipids. In contrast, regions exposed to the unstable stress pattern of turbulent blood flow show a more exaggerated thickening characterized by an abundance of smooth-muscle and connective-tissue cells, poorly oriented collagenous fibers, and a predilection for

lipid deposition. Plaques strikingly resembling lipid-laden

human lesions have been induced in rabbits, in the absence of special diets or hypercholesterolemia, by mechanical injuries from an implanted catheter. In these experiments, the morphology of the lesion depended on the relationship between the catheter and the arterial wall. Where repeated or continuous wall contact was likely, raised lipid-rich plaques developed, whereas in areas where lesser contact was probable, slightly elevated fibrous or pearly plaques

formed. Hypertension is an important endogenous source of mechanical stress known to accelerate atheroma forma¬ tion in experimental atherosclerosis. Its effect appears to be mediated by an increase in shearing forces on, and an overstretching of, the arterial wall that would promote endothelial damage and medial weakening. Not all sites of predilection for the development of atherosclerosis are accounted for entirely by local hemody¬ namic factors.11' There are segments of the arterial tree that are more susceptible than others, possibly because of differences in structural or metabolic features. In the dog made hypercholesterolemic, the abdominal segment of the aorta is more vulnerable to atherosclerosis than the thoracic segment. Interchange in the same animal of short lengths of thoracic and abdominal aorta showed that the transferred abdominal segment in the thorax still devel¬ oped more atherosclerosis than either the adjacent thoracic aorta or the thoracic segment that had been transferred to the abdominal location.2" A similar situation might exist in man, where coronary arteries and abdominal aorta are more susceptible to atheroma formation than the thoracic aorta. This difference in proneness has recently been ascribed to a difference in structure. Wolinsky and Glagov21 have identified a basic structural unit of the media made of concentric lamellar layers of elastin with subjacent smooth-muscle cells and scleroprotein fibers. These units are of fairly uniform composition and thick¬ ness from birth, regardless of species. Their number in mammalian arteries appears to be limited to about 29, unless medial vasa vasorum are present. The thoracic and abdominal segments of the aorta in man differ in both structure and supply of vasa vasorum. The thoracic aorta grows after birth by increasing its number of lamellar units to about 56, and the outer portion is supplied with vasa vasorum. In contrast, the media of the abdominal segment is virtually devoid of vasa vasorum and enlarges mostly by a widening of the existing 29 units. Thus, the human abdominal aortic wall is nourished mainly by transintimai perfusion (fewer than 29 units), despite the fact that its thickness greatly exceeds the "critical depth" of the avascular zone of most mammalian aortas with medial vasa vasorum. The relative absence of penetrating vasa vasorum in the coronary arteries and the abdominal aorta means that any interference with the delivery of oxygen and nutrient from the lumen to this avascular zone might compromise tissue respiration and nutrition and promote damage to the wall.

Immunological Injury

and Inflammation

In the long list of "endothelioclastic" means used to induce atherosclerosis in the experimental animal, much attention has been given in recent years to immunity and inflammation.22 The synergy of immunological injury (immune complex disease) and hypercholesterolemia has been demonstrated in the production of lesions resembling human atherosclerosis in several animal species, including the baboon. Antibodies directed against the arterial wall, lymphocytotoxic serum, or agents that activate the reticuloendothelial system have been shown to promote or enhance the development of atherosclerosis, but the mech¬ anisms involved are still a matter of debate. Proponents of the theory that there is an immunological start to athero-

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sclerosis believe that immune complexes become fixed in the vascular endothelium, which proliferates in response to the complement-mediated cytolytic attack. Histamine and other factors released locally increase permeability to cholesterol and other plasma constituents. Such a sequence repeated over many years would result in sizeable atheromatous lesions. Several elements in human pathology point to a possible role of immune factors in atherogenesis: extensive atherosclerotic lesions develop in the vessels of transplanted organs undergoing rejection; immune com¬ plexes (such as antibodies to milk proteins) reportedly circulate in the plasma of patients with coronary heart disease; autoantibodies against both normal and atheromatous aortas are found in human arterial disease; and immune complexes of lipoprotein and their antibodies are found in plasma as well as in atherosclerotic material obtained at surgery in an occasional hyperlipidemic

patient.

Inflammation may also alter endothelial permeability and arterial damage. Some clinical observations would be in accord with this possibility. Acute inflammatory changes have been reported in plaques from some cases of young coronary deaths. Appreciable aortic atherosclerosis has been found at autopsy in the arteries of young persons with a history of rheumatic fever. Atherosclerotic involve¬ ment tends to be more extensive and severe when it supervenes in various forms of arteritis such as temporal arteritis or Takayashi's disease. The possible involvement of immunity and inflammation in human atherogenesis remains an interesting hypothesis. Its exact nature and importance must await further studies. INTIMACYTE PROLIFERATION, PLATELETS, AND THROMBOSIS

Proliferation of intimai cells is the other major element in the pathogenesis of atherosclerosis (Fig 1). Most inves¬ tigators now agree that the intimacytes or myointimal cells are derived from smooth-muscle cells of the media. The medial cell can undergo all the transformations neces¬ sary to account for the various appearances of intimacytes seen in atherosclerosis. They have been grown in culture, where they can secrete both collagen and elastin. Choles¬ terol feeding induces a higher proliferative activity of the medial smooth-muscle cells near the internal elastic lamina adjoining intimai plaques. Stemerman and Ross23 showed by electron microscopy that the medial smooth-muscle cells appear to be crossing the internal elastic lamina through lacunae in early atherosclerotic lesions. The cells retain some of the morphological characteristics of smoothmuscle cells as they are gradually transformed into typical foam cells in the atherogenic process. From experiments carried out with aortas of cholesterolfed rabbits, de Duve24 has devised a model to account for the transformation of smooth-muscle cells into foam cells that is based on the inability of the lysosome to cope with the influx of cholesteryl esters into the cells. This author found that the lysosomes of aortic smooth-muscle cells of cholesterol-fed rabbits were less dense than those of normal animals, which suggested that they were accumu¬ lating lipids of low-density. He has also shown that the activity of cholesteryl esterase is very low in rabbit lysosomes. Thus, in the de Duve model, cholesteryl ester-

rich LDL

molecules, introduced into the intimai space alteration of endothelial permeability, are taken up by the smooth-muscle cells. The vesicles that carry the LDL molecules into the cell eventually fuse with the lysosome for storage and digestion. Since lysosomal cholesteryl esterase activity is low, and since cholesterol

through

some

must be released from its ester to be cleared from the

lysosome and be used by the cell or transported out of them, cholesteryl esters accumulate and the cells gradually become foamy. Several published observations are in line with these findings. In both human and experimental atherosclerosis, an unusually high number of lysosome-like structures have been reported. Lysosomes are also abnormally numerous in the aortic smooth-muscle cells of hypertensive vessels. Acid hydrolase activity is lower in vessels prone to atheroscle¬

rosis than in vessels that are more resistant. In the rhesus monkey, cholesterol and cholesteryl esters have been shown to accumulate early in the lysosomal fraction of well-differentiated smooth-muscle cells from experimental atherosclerotic lesions. The recent case report by Sloan and Fredrickson2S of massive aortic and coronary atheroscle¬ rosis in a young woman who was found to have very low acid cholesteryl esterase activity in her arterial tissues is another argument in favor of this. Proliferative Process and Growth Factors

Much of the information gained on the proliferative process in atherogenesis has been derived from experi¬ ments in animals and in tissue culture. These have been thoroughly reviewed by Ross and Glomset.2 When the arterial wall is selectively stripped of its endothelial lining, a series of events takes place that ultimately results in the repair of the injured vessel through a concerted action of

the platelets, the smooth-muscle cells of the inner media, and the endothelium. Stemerman and Ross23 conducted a critical experiment in Macaca nemestrina monkeys in which they followed up these events by electron microscopy after selective removal of the vascular endothelium by the passage of an intra-arterial balloon catheter through the iliac artery. Within minutes after injury, platelets adhere to the denuded subendothelial connective tissue, aggre¬ gate, and lose their granules. This platelet layer is grad¬ ually removed within 7 to 14 days as the vessel is progres¬ sively reendothelialized. Four to seven days after injury, smooth-muscle cells begin to undergo modification and appear to migrate through fenestrae in the internal elastic lamina into the intima, where they proliferate. Within 14 to 28 days, the proliferating intima may consist of five to ten layers of smooth-muscle cells surrounded by newly formed collagen fibrils, immature elastic fibers, and

proteoglycans.

After three months, the greatly hyperplastic lesion reaches a maximum thickness of about 15 layers of smoothmuscle cells with new extracellular connective-tissue elements. The endothelium is regenerated from the periph¬ ery, and it may take as long as nine months before it has regained a normal structure. As it regenerates, the smooth-muscle cells lesion regresses. After six months with no further injury, the lesion shrinks down to one or two layers of smooth-muscle cells in the normocholesterolemic animal. These lesions, which do not contain intracellular

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are similar to fibromusculoelastic lesions or preatherosclerotic intimai hyperplasia of man. A similar fundamental response of intimai smoothmuscle proliferation and formation of large amounts of connective-tissue protein has been observed after all forms of endothelial injury produced by a variety of noxious stimuli such as mechanical injury, homocystinemia, hypercholesterolemia, and immune injury. It appears that repeated injuries will maintain and exacerbate these lesions, and that hypercholesterolemia will promote their lipid infiltration, inducing changes resembling the more advanced form of atheromatous plaques seen in man. It has already been mentioned that repeated injury can produce lipid-laden atherosclerotic lesions, even when plasma cholesterol concentrations are normal. The sequence of events that follows balloon injury focuses attention on the endothelium as a barrier to the influence of the blood and its constituents on the proliferation of intimai smoothmuscle cells. It raises the possibility that platelets and plasma components are a source of "proliferating factors" acting on both the endothelium and the smooth-muscle cell. Indeed, arterial smooth-muscle cells in culture have been extensively used recently for the detection and study of such growth-promoting factors in plasma.1-'-'--" Serum LDL have clear growth-promoting properties; other lipoprotein fractions are much less active. Insulin in physiological concentrations appears to have a similar effect. The combined effects of lipoproteins and insulin, however, account for only part of the growth-promoting activity of serum. The major blood serum mitogen in such smoothmuscle cell cultures was found to be a factor released by platelets.26 This potent growth-stimulating factor is released by thrombin and is rather heat stable, nondialyzable, and apparently basic with a molecular weight of about 13,000. It has properties similar to those of the fibroblast growth factor isolated from bovine pituitary extracts by Gospodarowicz and Moran.27 It has been hy¬ pothesized that the factor could be produced by the pitui¬ tary and taken up and concentrated in platelet granules, to be released locally when needed. This would provide a unique means of transporting a pituitary polypeptide hormone to its target cell. This finding has given a new impetus to research on the role of platelets in atherogene¬ sis.

lipids,

Platelets

Historically, platelets have been linked to atherosclerosis through their role in thrombosis.5-28 The detection of fibrin in human plaques, the experimental demonstration that an induced thrombus could quickly evolve into a plaque, the observation that plasma lipoproteins are incorporated into organizing thrombi, the fact that in man, mural thrombi

far more common in the arterial circulation than occlusive thrombi, and other related observations lent support to the proponents of the encrustation theory. There now seems to be little doubt that thrombi can and do change into typical atherosclerotic lesions; what is far from being established is the relative importance of such a mechanism in the human disease. In recent years, new mechanisms have been evoked to account for the role of platelets in atherogenesis. On the one hand, this may are

depend on their ability to initiate a mural thrombus on an injured intimai lining, as discussed above, or on their ability to promote the proliferation of arterial smooth-

muscle cells in tissue culture. The sequence of events postulated is as follows: Injury to the endothelium, whether extensive or restricted, attracts platelets and provokes their adherence to the wall. During the process, they are stimulated to release their granule constituents, including adenosine diphosphate, which will cause platelets flowing past the injury site to change shape and adhere to each other, forming an aggregate that will further participate in the release reaction. The permeability of the area, collagen, and elastic tissue are altered by the substances released by the platelets. Smooth-muscle cells are made to proliferate by their exposure to growth-promoting factor released by platelets. As mentioned above, the ultimate outcome will depend on the degree, duration, and type of injury, as well as on the concomitant excess of LDL-cholesterol concentration in plasma. Platelet consumption at the site of injury is translated into a shortened platelet survival time and an increase in platelet turnover. In the experiment of Ross and Harker11 in monkeys that had been made hypercholesterolemic for 18 months and had lost by focal sloughing about 7% of their arterial endothelial surface, the platelet survival time was 6.2 days (8.0 days in control animals). When survival time is shorter, the platelets in circulation are on an average younger and adhere more readily to collagen. The prime importance of platelets in early ather¬ ogenesis is further emphasized by the observation that both dipyridamole (an antiplatelet aggregating agent capable of blocking the release reaction) and antibodies to platelets prevent the formation of plaques in response to

injury.2-7

Monoclonal

Hypothesis Despite its widespread support among investigators, the

response-to-injury hypothesis rests to date solely on animal experimentation. A major obstacle to extending animal studies to man is that initiating events in human athero¬ sclerosis are virtually impossible to identify and study. Recent experiments on human plaques obtained at autopsy has led to the hypothesis that plaques are, like benign tumors, formed by division of a single cell that has lost control of its growth: the monoclonal hypothesis (Fig. 3). Benditt and Benditt2" have applied to human arterial tissue (aorta and common iliac arteries) the technique that was first used to demonstrate the monoclonal origin of benign smooth-muscle cell tumors of the uterus. It is based on the generally accepted belief that only one of the two X

chromosomes in females expresses its genes because of the random inactivation of one of them during embryonic development. The gene that codes for glucose-6-phosphate dehydrogenase (G-6PD), an enzyme having two distinct forms (A and B isoenzymes), is carried on the X chromo¬ some. Cells of black women who are heterozygous for this gene will, because of the random inactivation of the X chromosome, synthesize at random one or the other form of G-6PD. A mixture of A and B isoenzymes is found in tissue extract from these women and is the expression of this cellular mosaicism. It was found that samples from nonatherosclerotic arterial wall media and intima from such

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BLACK FEMALE HETEROZYGOUS FOR G-6PD

NORMAL CELL OF

ARTERIAL WALL

MUTAGEN

refer to the

more comprehensive recent reviews on the subject17-21-24-30 for further information and for a more extensive bibliography.

Figure

1 is

reprinted

with

permission

from McGraw-Hill Book Co Inc.

-

ALTERED CELL IN POLYCLONAL

T

RANDOM INACT/VAT/ON OF ONE OF THE X CHROMOZOMES DURING EMBRYO DEVELOPMENT

ATHEROMATOUS PLAQUES

Dipyridamole-PersamiiMe. FACTOR

MONOCLONAL: ISOENZYME A OR

ISOENZYME B

LIPIO INFILTRATION ATHEROSCLEROSIS FOAM CELL FORMATION NECROSIS

Fig 3.—Monoclonal hypothesis. Taking advantage of fact that there is random inactivation of one of X chromosomes coding for

glucose 6-phosphate dehydrogenase (G-6PD) during embryogenesis (left), Benditt and Benditt29 measured G-6PD isoenzymes A and B in arterial wall samples obtained at autopsy in women heterozygous for G-6PD. They found expected cellular mosaicism in normal aorta, but only one cell type (either A or B) in nearby atheromatous plaques (center). They proposed that some cells of arterial wall are altered by mutagens (hydrocarbons, viruses, etc) during life and develop selective advantage for proliferation. When exposed to promoting factors such as hypertension or hypercholesterolemia, these cells, kept in a subthreshold neoplastic state, are made to proliferate at a higher rate than surrounding cells and lead to formation of an atheromatous plaque (right). showed the expected mosaicism. In contrast, fibrous caps, even of relatively large athero¬ matous plaques, were composed of cells that produced solely or predominantly one enzyme type (either A or B). These results are compatible with a monoclonal (or monotypic) origin of human atheromatous plaques. Other inves¬ tigators have confirmed these findings and have further shown that fatty streaks, unlike fibrous plaques, did not show this proliferation of a single cell line.™ It has been suggested that each lesion is a benign neoplasm derived from a mutant cell that has developed selective advantage and has been transformed by such agents as chemicals (eg, cigarette-derived hydrocarbons, carcinogens of dietary origin) or viruses. Their potential for multiplying could be encouraged by such promoting factors as hypertension and hypercholesterolemia. Although the interpretation of these findings is a matter of much debate,2 the monoclonal hypothesis does represent a new departure in the study of atherosclerosis. It demands réévaluation of our present women

knowledge.

This brief review has attempted to draw attention to of the major gains in knowledge and new theories that have been acquired in the past few years and are currently guiding atherosclerosis research. I have insisted on the key elements that remain at the center of the atherogenic process: lipoproteins, endothelial injury, platelets, and smooth-muscle cell proliferation. Because of space limitations, I have been forced to leave out many interesting and pertinent observations and many chal¬ lenging new hypotheses. I urge the interested reader to some

Name and Trademark of Drug

Nonproprietary

-PROMOTING

EXCESSIVE MONOCLONAL PROLIFERATION

./

CELLULAR MOSAICISM 2 ISOENZYMES

"SUBTHRESHOLD STATE"

NEOPLASTIC

References 1. Davignon J: Current views on the etiology and pathogenesis of atherosclerosis, in Genest J, Koiw E, K\l=u"\chelO (eds): Hypertension: Physiopathology and Treatment. New York, McGraw-Hill Book Co Inc, 1977,

pp 961-989. 2. Ross R, Glomset JA: The pathogenesis of atherosclerosis. N Engl J Med 295:369-377, 420-425, 1976. 3. Walton KW: Pathogenetic mechanisms in atherosclerosis. Am J Cardiol 35:542-558, 1975. 4. Wissler RW: Development of the atherosclerotic plaque, in Braunwald E (ed): The Myocardium: Failure and Infarction. New York, Hospital Practice Publishing Co, 1974, pp 155-166. 5. Haust MD: Platelets, thrombosis and arteriosclerosis, in Hirsch J (ed): Platelets, Drugs and Thrombosis. Basel, Switzerland, S. Karger, 1975, pp 94\x=req-\ 110. 6. Marx JL: Atherosclerosis: the cholesterol connection. Science 194:711\x=req-\ 755, 1976. 7. Kolata GB: Atherosclerotic plaques: Competing theories guide research. Science 194:592-594, 1976. 8. Stehbens WE: The role of lipid in the pathogenesis of atherosclerosis. Lancet 1:724-727, 1975. 9. Armstrong ML: Evidence of regression of atherosclerosis in primates and man. Postgrad Med J 52:456-461, 1976. 10. Barndt R, Blankenhorn DH, Crawford, DW, et al: Regression and progression of early femoral atherosclerosis in treated hyperlipoproteinemic patients. Ann Intern Med 86:139-146, 1977. 11. Ross R, Harker L: Hyperlipidemia and atherosclerosis. Science 193:1094-1100, 1976. 12. Brown MS, Goldstein JL: Receptor-mediated control of cholesterol metabolism. Science 191:150-154, 1976. 13. Goldstein JL, Brown MS: Lipoprotein receptors, cholesterol metabolism and atherosclerosis. Arch Pathol 99:181-184, 1975. 14. Miller GJ, Miller NE: Plasma-high-density-lipoprotein concentration and development of ischaemic heart disease. Lancet 1975. 15. Heyden S: The problem with triglycerides. Nutr Metab 18:1-5, 1975. 16. Zilversmit DB: Mechanisms of cholesterol accumulation in the arterial wall. Am J Cardiol 35:559-566, 1975. 17. Bell FP, Day AJ, Gent M, et al: Differing patterns of cholesterol accumulation and 3H-cholesterol influx in areas of the cholesterol-fed pig aorta identified by Evans blue dye. Exp Mol Pathol 22:366-375, 1975. 18. Fry DL: Certain chemorheologic considerations regarding the blood vascular interface, with particular reference to coronary artery disease. Circulation 39(suppl 4):38-57, 1969. 19. Glagov S: Mechanical stresses on vessels and the non-uniform distribution of atherosclerosis. Med Clin North Am 57:63-77, 1973. 20. Haimovici H, Maier N: Role of arterial tissue susceptibility in experimental canine atherosclerosis. J Atheroscler Res 6:62-74, 1966. 21. Wolinsky H, Glagov S: Nature of species differences in the medial distribution of aortic vasa vasorum. Circ Res 20:409-421, 1967. 22. Poston RN, Davies DF: Immunity and inflammation in the pathogenesis of atherosclerosis. Atherosclerosis 19:353-367, 1974. 23. Stemerman MB, Ross R: Experimental arteriosclerosis: I. Fibrous plaque formation in primates, an electron microscope study. J Exp Med 136:769-789, 1972. 24. Duve C de: The participation of lysosomes in the transformation of smooth muscle cells to foamy cells in the aorta of cholesterol-fed rabbits. Acta Cardiol [Suppl] 20:9-25, 1974. 25. Sloan HR, Fredrickson DS: Enzyme deficiency in cholesteryl-ester storage disease. J Clin Invest 51:1923-1926, 1972. 26. Ross R, Glomset J, Kariya B, et al: A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Nat Acad Sci USA 71:1207-1210, 1974. 27. Gospodarowicz D, Moran JS: Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts. J Cell Biol 66:451-457, 1975. 28. Gresham GA: Early events in atherogenesis. Lancet 1:614-615, 1975. 29. Benditt EP, Benditt JM: Evidence for a monoclonal origin of human atherosclerotic plaques. Proc Nat Acad Sci USA 70:1753-1756, 1973. 30. Pearson TA, Wang A, Salezo K, et al: Clonal characteristics of fibrous plaques and fatty streaks from human aortas. Am J Pathol 81:379-387, 1975.

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