Acta Med Scand, Suppl. 715: 33-38

Growth Factors in the Pathogenesis of Atherosclerosis

RUSSELL ROSS From the Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA

Although the advanced lesions of atherosclerosis have been clearly established as smooth muscle proliferative lesions, the basis for the focal migration and proliferation of smooth muscle cells at particular anatomic sites in the cardiovascular system has never been well explained. The details presented in this paper demonstrate our ability to definitively identify the constituents of the advanced lesions of atherosclerosis using monoclonal antibodies. A detailed chronological study of the cellular interactions that occur at each stage of chronic hypercholesterolemia has permitted us to understand the nature of the cellular interactions that occur, and which precede the development of each phase of the lesions of atherosclerosis. The discovery of numerous growth factors, including platelet-derived growth factors (PDGF), a mitogen for connective-tissue-forming cells; epidermal growth factor (EGF) , a mitogen for epithelial cells and some connective-tissue-forming cells; fibroblast growth factor (FGF) , an angiogenic agent and mitogen for some connective-tissue-forming cells; and transforming growth factor-beta (TGFB), a factor that can act either as a mitogen or an inhibitor of cell proliferation, has permitted further examination of the cells respon- sible for the formation of these factors, the stimulus for their formation, and their possible roles in atherosclerosis.

The lesions of atherosclerosis The lesions of atherosclerosis take essentially two forms. These are the ubiquitous fatty streak and the advanced lesion or fibrous plaque (1,2). The fatty streak is found throughout life, but may occur as early as infancy, is quite common in children, and continues through- out life to be found in elderly individuals. The advanced lesion, or fibrous plaque, can also be obseved in young individuals such as those with some genetic forms of hyperlipidemia, but is generally found in increased frequency with increasing age. When the fibrous plaque progresses, it can expand and compromise the arterial flow. When this occurs, it can lead to myocardial infarction and cerebral infarction (3).

Numerous risk factors have been associated with increased incidence of the lesions of atherosclerosis; however, the most common relationship among such factors in Western civili- zation is that held between chronic hyperlipidemia and hypercholesterolemia, and this dis- ease process. Other risk factors that are closely correlated with atherosclerosis include dia- betes, cigarette smoking, hypertension, and increasing age (4). Characteristically, the lesions of atherosclerosis have three dominant biological characteristics. These are 1) proliferation of smooth muscle cells which accumulate together with variable but large numbers of blood- monocyte-derived macrophages; 2) large amounts of connective tissue matrix proteins in- cluding collagen, elastic fibers, and proteoglycans that surround the smooth muscle cells re- sponsible for their formation; and 3) lipid accumulation in the form of foam cells, within smooth muscle cells and macrophages, and as deposits within the extracellular matrix sur- rounding these cells.

Monoclonal antibodies demonstrate the cellular constituents Although numerous studies have described smooth muscle cells and macrophages as impor- tant components of the lesions of atherosclerosis, the standard histologic techniques avail-

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34 R. Ross Acta Med Scand, Suppl. 715

able for analyzing the morphology of the lesions often do not provide sufficient resolution to determine cell type. Recently it has been possible to obtain monoclonal antibodies specific for smooth muscle cells (S), for macrophages (6) , for various forms of lymphocytes, and for endothelial cells. Using these monoclonal antibodies, we have been able to demonstrate that smooth muscle cells and macrophages are ubiquitous and are present in large numbers in the advanced lesions of atherosclerosis. In contrast, the earliest lesion of atherosclerosis, the fatty streak, consists initially of essentially pure populations of lipid-laden macrophages. With increasing age, the fatty streak begins to accumulate lipid-laden smooth muscle cells which enter the intima of the artery beneath the foamy macrophages (2).

Studies in chronically hypercholesterolemic nonhuman primates (7 ,8) , swine (9) , and rab- bits (10) all demonstrate that the advanced lesion, or fibrous plaque, appears to form as a re- sult of progression of fatty streaks that continue to accumulate both smooth muscle cells and macrophages and become converted to classical fibrous plaques (11). At some point during their development, many of the fibrous plaques also contain numerous lymphocytes (12). The lesions of atherosclerosis take somewhat different forms, depending upon the artery in which they are located and the associated risk factors. In hypercholesterolemic individuals, they are invariably lipid-laden, whereas in cigarette smokers or hypertensives, many of the lesions may be very fibrous and contain little apparent lipid (13).

The cellular events in hypercholesterolemia Faggiotto et al. (7) studied the sequence of events that occur in a large series of pigtail mon- keys fed a high-fat, high-cholesterol diet. These animals were maintained at cholesterol and plasma LDL levels equivalent to those found in homozygous human familial hypercholes- terolemic individuals (500-1 200 mg/dl).

Under these circumstances, the first changes were observed within a week of commencing a hypercholesterolemic regimen, when randomly localized clusters of monocytes attached to the endothelium throughout the arterial tree. These monocytes appeared to migrate on the surface, pass between endothelial cells, and localize subendothelially. Within relatively short periods of time, these monocytes became activated as macrophages, taking up large quan- tities of lipids and acting as scavenger cells during this process. They eventually became lipid- laden foam cells and changed the smooth surface of the overlying endothelium to an irregu- lar surface, raised up by the continual accumulation of lipid within the cells and the accumu- lation of macrophages. With time, the fatty streaks were seen to expand not only due to mac- rophage accumulation, but because smooth muscle cells migrate from the media of the artery into the intima and also take up lipid to become foam cells.

After four to five months on the hypercholesterolemic regimen, a series of secondary changes occur in many of these fatty streaks at particular anatomic sites such as branches and bifurcations. These manifest as separations between junctions of endothelial cells that over- lie fatty streaks at these sites, leading to endothelial retraction and exposure of the underly- ing foam cells or macrophages. In many instances, the foam cells appear to be swept into the circulation. In others, they are covered by mural thrombi of platelets. Where the foam cells are lost, platelets often adhere to the exposed subendothelial connective tissue. After one to two months time, wherever platelets adhere and form mural thrombi, similar anatomic sites become changed into extensive smooth muscle proliferative fibrous lesions. These observa- tions suggest that wherever platelet interactions occur, they are relatively rapidly followed by smooth muscle proliferative lesions.

In numerous instances, however, the endothelium remains intact and no platelet interac- tions occur. At these sites, over a somewhat longer period of perhaps four to six months, ad- vanced proliferative lesions of atherosclerosis also occur. These lesions appear to take longer to form, but are eventually similar in appearance to the more rapidly forming lesions. In each

Acta Med Scand. Suppl. 715 Growth factors and atherosclerosis 35

of these cases, the lesions are covered by a dense fibrous cap consisting largely of smooth muscle cells surrounded by large amounts of dense connective tissue matrix, together with randomly dispersed monocytes, macrophages, and lymphocytes. These fibrous caps overlie a very cellular component of the lesion that contains numerous smooth muscle cells and mac- rophages, many of which contain lipid deposits. Beneath this cellular region, there may be areas of necrosis, cell debris, and extracellular lipid.

A virtually identical sequence of events has been observed by Rosenfeld et al. (10) in the Watanabe Heritable Hyperlipidemic (WHHL) rabbit (the only available animal model for homozygous human familial hypercholesterolemia) and in the fat-fed rabbit. Their observa- tions also suggest that the fatty streak, which consists initially of activated monocyte-derived macrophages, can progress through a variety of changes to the proliferative advanced lesion of atherosclerosis, the fibrous plaque. This raises the interesting question as to how these pro- liferative smooth muscle lesions occur.

Growth factors and smooth muscle proliferation Since the discovery that a growth factor derived from activated platelets, platelet-derived growth factor (PDGF), is the principal mitogen in whole blood serum responsible for the growth of many cells in cell culture (14, 15), it has been shown that numerous other cells can also serve as sources of similar if not identical growth factors. In addition to PDGF, many other growth factors are also formed and secreted by different cells, including fibroblast growth factor (FGF), a potent angiogenic agent; epidermal growth factor or transforming growth factor alpha (EGF or TGF-a), potent mitogens, particularly for epithelium; and transforming growth factor beta (TGF-p), a factor that can be an inhibitor or an agonist of proliferation, depending upon the circumstances.

For example, when platelets are exposed to thrombin, collagen, or ADP, they release four growth factors: PDGF, FGF, TGF-a (or EGF), and TGF-8. The same is true for the activated macrophage (16). Furthermore, it has been shown that appropriately activated endothelial cells can secrete a form of PDGF as well as another growth factor (17). In some cases, it has also been demonstrated that rat arterial smooth muscle cells from newborn (in contrast to adult) rats appear to be capable of secreting a PDGF-like molecule in culture (18). Further- more, smooth muscle cells from catheter-induced proliferative lesions in the rat carotid also secrete a form of PDGF (19). With the development of the techniques of molecular biology, it is possible with the use of appropriate cDNA probes to demonstrate the appearance of genetic expression in the cells, leading to messenger RNA formation in the cells as they are stimulated to form these growth factors (16).

Thus with appropriate changes in the artery wall and with appropriate stimulation, all of the cells known to be involved in the process of atherogenesis can potentially serve as potent sources of growth factors for smooth muscle cells. When these cells proliferate, they may also be stimulated to secrete new connective tissue molecules, to increase their uptake of low- density lipoprotein, and potentially to become foam cells.

Platelet-derived growth factor PDGF is an extraordinarily potent mitogen. It binds to susceptible cells such as smooth mus- cle or fibroblasts with very high affinity (kD approx. lo-'' M), and upon doing so stimulates the cells to undergo many changes (20). Some of these effects occur very rapidly, including changes in the receptor for PDGF that lead to a sequence of intracellular events which cul- minate in synthesis of new DNA and thus cell proliferation. In addition, PDGF is a potent chemotactic agent that has the capacity to specifically attract cells to migrate in a concentra- tion gradient of PDGF, and thus may potentially serve as an important means of attracting smooth muscle cells from the media into the intima of the artery if appropriate endothelial

36 R. Ross Acta Med Scand, Suppl. 715

Fig. 1. The responses to injury hypothesis. Advanced intimal proliferative lesions of atherosclerosis may occur by at least two pathways. The pathway demonstrated by the clockwise (long) arrows to the right has been observed in experimentally induced hypercholesterolemia. Injury to the endothelium (A) may induce growth factor secretion (short arrow). Monocytes attach to endothelium (B), which may continue to secrete growth factors (short arrow). Subendothelial migration of monocytes ( C ) may lead to fatty streak formation and release of growth factors such as PDGF (short arrow). Fatty streaks may become directly converted to fibrous plaque (long arrow from C to F) through release of growth factors from macrophages or endothelial cells or both. Macrophages may also stimulate or injure the overlying en- dothelium. In some cases, macrophages may lose their endothelial cover and platelet attachment may occur (D), providing three possible sources of growth factors-platelets, macrophages, and endothelium (short arrows). Some of the smooth muscle cells in the proliferative lesion itself (F) may form and sec- rete growth factors such as PDGF (short arrows).

An alternative pathway for development of advanced lesions of atherosclerosis is shown by the arrows from A to E to E In this case, the endothelium may be injured but remain intact. Increased endothelial turnover may result in growth factor formation by endothelial cells (A). This may stimulate migration of smooth muscle cells from the media into the intima, accompanied by endogenous production of PDGF by smooth muscle as well as growth factor secretion from the injured endothelial cells (E). These in- teractions could then lead to fibrous plaque formation and further lesion progression (F). Reproduced by permission ofThe New England Journal of Medicine (Ross R. The pathogenesis of atherosclerosis- an update. N Engl J Med 1986; 314: 496.)

Acta Med Scand, Suppl. 715 Growth factors and atherosclerosis 37

injury has occurred and PDGF is deposited (21). PDGF can also induce increased binding of LDL to smooth muscle cells, increased cholesterol synthesis, and a host of other intracel- Mar changes (22, 23).

The response to injury hypothesis of atherosclerosis Taken together, the observations described in this paper support a hypothesis that proposes that some form of injury may occur to the endothelium in susceptible individuals who are exposed to agents that can initiate atherogenesis. Hypercholesterolemia is one of the most prominent of these and can lead, as noted above, to changes in circulating monocytes and in endothelium in the artery, resulting in increased monocyte attachment. These changes in the endothelium may also lead to increased endothelial cell turnover, and potentially to expres- sion of the gene for platelet-derived growth factor in the injured endothelial cells. It fol- lows that perhaps one of the earliest forms of injury may result in formation and secretion of growth factors, including PDGF, by the activated endothelial cells. Monocyte attachment is followed by subendothelial migration of the monocytes and their conversion to macro- phages. Such macrophage formation could then lead to formation and secretion of growth factors including PDGF, FGF, EGF-like and TGF-P by the activated macrophages. These two cellular responses would be sufficient to attract smooth muscle cells into the intima of the artery and to stimulate their proliferation and formation of connective tissue. Thus fatty streak formation, which would include activated macrophages, would be sufficient to lead to advanced lesion formation if the activated cells were capable of forming and secreting growth factors ( 2 ) .

Furthermore, the response to injury hypothesis suggests that if the injury continues and platelet interactions occur, the platelet together with the activated endothelium and macro- phages could provide even larger amounts of the different growth factors and could further enhance lesion formation and progression. This is described in Fig. 1.

It is important to note that advanced lesions of atherosclerosis can form both when the en- dothelium remains intact and when endothelial retraction occurs. In the former case, en- dothelium and macrophages could serve as the source of growth factors; in the latter, platelets could also act together with these two cells to play this role (2).

CONCLUSIONS

It can be seen that changes in the endothelium leading to injury of the endothelial cells may in themselves be sufficient to generate a sequence of events that can lead to smooth muscle proliferation, lipid accumulation, and formation of new connective tissue. Together, these events may result in the formation of advanced proliferative lesions of atherosclerosis, which commence as fatty streaks and then progress to become fibrous plaques. Maintenance of endothelial homeostasis and prevention of endothelial injury are therefore potentially im- portant components in the prevention of atherosclerosis. On the other hand, it is also con- ceivable that it may be possible to intervene at the level of monocytehacrophage interac- tions with the endothelium, with macrophage formation, and ultimately with the formation and secretion of growth factors by activated endothelium and/or macrophages. Finally, in some cases it may be possible for proliferating smooth muscle cells to form growth factors themselves. If this is true in other species besides the rat, it would be important to find means of preventing smooth muscle cell formation of growth factors as well. These studies suggest new approaches to understanding how the process of atherogenesis occurs and the stages of lesion development at which it may be possible to intervene. This may lead to the develop- ment of specific agents that interfere with the formation and release of growth factors which prompt cellular changes, and thus potentially with atherogenesis.

38 R. Ross Acta Med Scand, Suppl . 715

ACKNOWLEDGEMENTS The work reported here was supported in part by grants from NHLBI, HL-18645; by a grant to the Re- gional Primate Center, University of Washington, RR-00166; and by a grant from RJR Nabisco, Inc.

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Correspondence: Russell Ross, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195, USA.