Common mechanisms of proliferation of smooth muscle in atherosclerosis and hypertension

Common Mechanisms of Proliferation of Smooth Muscle in Atherosclerosis and Hypertension STEPHEN M. SCHWARTZ, MD, AND MICHAEL A. REIDY,PHD At least two exogenous sources o f agents able to control vascular smooth muscle proliferation can be identified. Platelets contain and release mitogens as well as a factor, TGF-[3, that inhibits cell growth on plastic surfaces while stimulating it when cells are grown in suspension in soft agar. Macrophages release mitogens, including PGDF, and macrophage invasion is characteristic of early experimental lesions in fat-fed animals. Finally, it is at least possible that endothelial cell production of mitogens may represent a response to some as yet undefined external injury. The vessel wall also offers sources of growth control endogenous to the smooth muscle cell layers. The vessel wall contains heparan sulfate able to inhibit cell growth of smooth muscle cells, which by themselves can synthesize PDGF. This provides possible positive and negative control of replication intrinsic to the smooth muscle cells themselves. The role of these intrinsic or extrinsic factors in the smooth muscle proliferation of hypertension and atherosclerosis remains hypothetical. It is intriguing to implicate platelets and/or macrophages in the denuding injuries seen in small hypertensive vessels and in advancing atherosclerotic plaques. At least for the latter case, however, there seem to be other critical factors. Simple denudation and thrombosis, for example, are not sufficient to stimulate smooth muscle growth, and the kinetics of proliferation after balloon denudation imply the presence of some other event required to initiate smooth muscle proliferation. Similarly, smooth muscle replication in large vessels of hypertensive animals occurs without loss of endothelial continuity. This implies that replication in response to hypertension depends on factors intrinsic to the vessel wall. Benditt's observation of monoclonality also implies some intrinsic mechanism allowing cells to grow in a focal manner. It is intriguing to consider the possibility that this commitment process could require the release of cells from the intrinsic inhibitory effects of heparan sulfate located around the cells or the synthesis of growth factors secreted by the smooth muscle cells themselves. If we add the hypothesis that only some cells are capable of such a response, we would expect the sort of oligodense phenomenon demonstrated by Benditt. Proof of such a hypothesis, however, will have to await development of methods to explore these mechanisms directly in the vessel wall responding to injury. HUM PATHOL18:240--247, 1987. I n all tissues, scarring consists o f cell proliferation a n d deposition o f connective tissue. By this definition, the lesions o f both atherosclerosis a n d h y p e r tension could be d e f i n e d as sclerosis o r scarring o f arterial tissue, l This is an apt description, because b o t h diseases involve s m o o t h muscle p r o l i f e r a t i o n a n d connective tissue f o r m a t i o n . T h e r e is evidence that the proliferative aspect o f Received from the Department of Pathology, University of Washington, Seattle, Washington. Address correspondence and reprint requests to Dr. Schwartz: Professor of Pathology, University of Washington, Seattle WA 98195. 0046-8177/87 $0.00 + .25

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sclerosis is an essential part o f the vascular pathology o f both h y p e r t e n s i o n a n d atherosclerosis. For example, retrospective anatomic studies o f the distribution o f lesions in m a n show a high c o r r e l a t i o n between localization o f classical lesions in the adult and focal distribution o f intimal cell masses in the very y o u n g . 2 We have suggested that these focal masses could a c c o u n t f o r the oligo-clonal n a t u r e o f adult a t h e r o s c l e r o t i c lesions. This w o u l d imply that cell proliferation may be the initial event in lesion formation. 3 Similarly, s m o o t h muscle proliferation is a rem a r k a b l e p a r t o f the p a t h o l o g y o f h y p e r t e n s i o n . Folkow has p r o p o s e d that increased structural mass m a y u n d e r l y the i n c r e a s e d p e r i p h e r a l resistance characteristic o f even early hypertension. Cytokinetic and m o r p h o m e t r i c studies suggest that the bulk o f this increase is in the f o r m o f new cells. 4-7 B e f o r e e x t e n d i n g the role o f s m o o t h muscle proliferation too far, we must consider briefly the differences in the lesions elicited in h y p e r t e n s i o n and atherosclerosis. Atherosclerosis occurs in large elastic arteries a n d m e d i u m - s i z e d muscular arteries. T h e s e vessels b e c o m e occluded by lesions that enlarge because o f proliferation o f s m o o t h muscle, lipid accumulation, a n d thrombosis, s Hypertensive lesions are usually described in pathology texts as o c c u r r i n g in the m e d i a a n d intima o f small arteries, the "resistance vessels." H o w e v e r , large vessels a r e affected also. 4,6 While lipid accnmulation is not part o f the pathology o f h y p e r t e n s i o n , it is true that h y p e r t e n s i o n accelerates lipid accumulation in large vessels. 9 T h u s , from the pathologic point o f view, the lesions caused by the two diseases are similar and both involve abnormal smooth muscle proliferation. Differences have to do with the affected vessels a n d secondary changes. T h e question for this review is w h e t h e r we can identify the basic mechanisms u n d e r l y i n g these proliferative responses.

LESIONS OF ATHEROSCLEROSIS A fully d e v e l o p e d but uncomplicated atherosclerotic lesion has several c o m p o n e n t s . T h e s e include proliferated s m o o t h muscle cells, lipid, necrotic cells, a n d c o n n e c t i v e tissue. T h e s e c o m p o n e n t s are situated in a specific pattern. Beneath the e n d o t h e l i u m , there is a layer o f dense, fibrous, connective tissue. T h i s l a y e r , c a l l e d a f i b r o u s cap, c o n t a i n s m a n y s m o o t h m u s c l e cells a n d c o v e r s the fatty mass o f " a t h e r o m a . " T h e o t h e r major a n d characteristic cell o f the lesion is the m a c r o p h a g e . M a c r o p h a g e s are

VASCULARSMOOTHMUSCLEPROLIFERATION[Schwartz & Reidy]

usually found between the endothelium and the fibrous cap and adjacent to the central fatty mass. At least two d i f f e r e n t lesions have been suggested as morphologic precursors of this classical lesion. One of these lesions is simply a focal accumulation of smooth muscle cells in the tunica intima. The arterial wall is made up largely of smooth muscle cells in the layers o f the mnica media. Some cells, however, become entrapped within the intima during development. T h e number of these cells increases with age in a pattern sometimes called diffuse intimal thickening. 1~Large masses of such cells are seen near vascular b r a n c h e s in s t r u c t u r e s called i n t i m a l c u s h i o n s . 11,12 M a n y p a t h o l o g i s t s r e g a r d t h e s e cushions as n o r m a l structures. Studies o f the response to fat feeding, as well as studies of the localization of atherosclerotic lesions in man, however, show a coincidence between location of mature atherosclerotic lesions of the sort described here and the presence, i n the fetus and young animal, of intimal cell masses, ll'12 T h e Velicans have suggested that these intimal cell masses in children transform into the atherosclerotic lesions of adults. A more traditional idea is that atherosclerotic lesions begin with fatty accumulation. T h e earliest lesion would be a lesion called a fatty streak. 13 This structure is usually defined by its gross anatomy. Focal accumulation o f large amotmts o f lipid are easily visible through the intimal side of the vessel. Most obvious o f the early lesions, the fatty streak probably represents a focal accumulation of foam cells, ceils with many lipid droplets in their cytoplasm. These may be derived from macrophages or smooth muscle cells. The most important reason for considering the fatty streak as the early lesion of atherosclerosis is the ability of cholesterol-rich diets to induce foam-cell lesions that later progress to form the classical lesion described here. It is likely that this sort of process in experimental animals occurs in humans as well. Fatty streaks are found in infants, suggesting a condition with early exposure to lipids in milk. Whether formation of fatty streaks occurs in preexisting intimal cell masses and whether intimal cell masses can progress to form lesions without a foam-cell stage remain unexplored questions. It is important for this discussion, however, to consider the process of the formation of the atherosclerotic lesion as a lifelong process. LESIONS OF HYPERTENSION As we have already noted, hypertension produces smooth muscle proliferation in all arterial beds. T h e significance of this finding for hypertension itself is unknown. Smooth muscle proliferation in large vessels should have little effect on blood pressure, because large vessels do not contribute to resistance to flow. Pathology of smaller arteries in hypertension obviously is more important. Unfortunately, we do not know to what extent 241

the changes in small vessels represent a response to high blood pressure versus the possible role of these morphologic changes in the etiology o f high blood pressure. Physiologic studies of all forms of chronic hypertension in humans and animals show that small vessels have increased resistance to flow even when vascular tone is relaxed completely. This resistance to flow, as proposed by Bjorn Folkow,4 must be structural, because it persists even when the active contractile force of the smooth muscle cell has been ablated. T h e significance of the structural component of hypertension in humans is difficult to determine. T h e effectiveness of antihypertensive agents would argue that active contractile force is more important. Nevertheless, minute changes in the dimensions of the lumen can account for large increases in resistance. Folkow4 estimates that a 5 per cent reduction in lumen, a variation too small to be measured by the pathologist, should produce a 20-mm increase in blood pressure. Moreover, the role of the contractile mass in active maintenance of pressure should be considered. Morphometric studies show more contractile apparatus as well as more extracellular matrix. 14 This increase in medial smooth muscle mass also differs from changes in atherosclerosis because it contributes to the function of the vessel wall, that is, its ability to contract. Unlike atherosclerosis, hypertension produces an increase in the mass, or the n u m b e r , o f smooth muscle cells without cansing those cells to migrate into the tunica intima. It may be correct to refer to hypertensive atherosclerosis as an adaptive change that increases the ability of the vessel wall to maintain an elevated pressure. Conversely, we might also imagine this "adaptive" change itself becoming a cause of the maintenance of elevated peripheral resistance. T h e cellular basis for increased mass contributing to peripheral resistance may be quite simple. Hypertension itself may produce a variety of adaptive c h a n g e s i n c l u d i n g alterations in m e m b r a n e properties, salt content, and connective tissue synthesis. All of these alterations, however, are readily reversible. Unless cell death occurs, changes in DNA are permanent. Acute hypertension produces proliferation o f smooth muscle cells in arteries of all sizes. In the aorta this change is irreversible, even following antihypertensive therapy. 15.16 Moreover, the change in the aorta seems particularly adaptive, because DNA synthesis occurs without cytokinesis. That is, cells replicate their DNA without dividing; instead, they form tetraploid, octaploid, or even hexadecaploid cells. 17 T h e altered s u r f a c e - v o l u m e - D N A ratios of the polyploid cells could account for some of the many reports of altered physiologic responsiveness of smooth muscle cells in the aorta o f hypertensive animals. Ls More importantly, the ability to induce such changes implies that even a brief hypertensive episode may leave the arterial tree with a permanent memory and, possibly, a permanent alteration in responsiveness to external stimuli.

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ROLE OF HYPERTENSION IN ATHEROGENESIS O u r discussion of atherosclerosis thus far focused on the role of smooth muscle cells, lipid, and thrombosis in contributing to the mass o f the lesion. The most obvious mechanism for hypertension as a risk factor is the possibility that hypertension exerts increased pressure on the vessel wall, causing rapid filtration and uptake o f plasma proteins into the vessel wall. In the present context, however, we need to consider the possible role of changes in permeability in hypertension in stimulating the kind o f smooth muscle proliferation seen in atherosclerosis. Unfortunately, it is not clear why permeability is increased in hypertension. Ultrastructurally, the endothelial cell appears as a continuous cell layer. Normal endothelial cell junctions appear to be closed by intermembranous complexes that should prevent the passage of lipoproteins between the cells. 19-~l In acute hypertension these junctions break down, allowing thrombosis and access of plasma and plateletderived proteins to the underlying vessel wall. 22 In contrast, chronic hypertension shows an increase in the complexity of endothelial cell tight junctions, if it shows any change. Since there are no continuous channels or pores across aortic endothelium, 23 any increase in transport must be attributed to vesicular transport. 24 The problem with these changes in permeability as a mechanism for eliciting growth is that, as yet, we have no evidence that plasma contains mitogens. 25 Thus, alterations in permeability of hypertensive vessels might accelerate lipid accumulation, but there is no reason to expect an increased rate of smooth muscIe proliferation. We need then to consider the 9role of more serious disruptions of the endothelium, that is, denuding injuries. ROLE OF DENUDATION I f loss of integrity of endothelium, as proposed by Ross and Harker in 1976, s,26 is a critical event in stimulating smooth muscle proliferation; when does it occur? Is this a critical initial event? Does it elicit rapid growth of smooth muscle cells? It is difficult to answer these questions. T h e concept o f endothelial continuity in large vessels assumed particular importance as a result of the postulated relationship between endothelial injury and the d e v e l o p m e n t o f atherosclerosis. 27.2s Several researchers have used catheters to remove intimal tissue, mainly endothelial cells, from the aortas o f rabbits, rats, and monkeys. When these injuries were extensive, the arterial wall uniformly responded by cell proliferation, fiber synthesis, and lipid accumulation.29-34 An explanation of the proliferation o f smooth muscle cells in response to endothelial denudation came from the work of Ross and coworkers. They found that platelets release a factor that promotes 242

the proliferation o f smooth muscle cells and other mesenchymal cells. 35-37 When the. endothelium is removed, platelets adhere to the subendothelial collagen s~ and release the contents of the granules, including the platelet-derived growth factor (PDGF), ~s as well as other, as yet, poorly defined polypeptide m i t o g e n s ) 9 PDGF is a polypeptide with a defined structure 4~ and a characterized receptor. 43,44 The receptor-ligand interaction results in the activation of a protein kinase that selectively phosphorylates tyrosine residues of membrane proteins in a manner similar to the event described for another mitogen, epidermal growth factor. 45 PDGF also stimulates cell migration, the other feature of smooth muscle response to injury in vivo.46 From our point of view, the critical issue in this model is the requirement for denuding endothelial injury. It is clear that PDGF is important in the repair process after tissue injury when tile injury is accompanied by vessel damage and platelet aggregation. However, it still remains to be demonstrated that endothelial denudation occurs prior to the formation of atherosclerotic lesions. That is, it is not certain that denudation is an initiating event, ahhough there is considerable evidence for platelet aggregation and thrombus formation on advanced lesions. Moreover, studies of carefully made endothelial wounds 47 and the kinetics of cell replication following the balloon catheter 4s show that denudation by itself, even with thrombosis, is insufficient to initiate replication and smooth muscle replication continues for days and weeks after completion of the bulk of the thrombotic response to endothelial denudation by the catheter. It is also important to point out that denudation is not an early event in experimental atherosclerosis. Absence of evidence for naturally occurring endothelial d e n u d a t i o n could be due to technical difficulties; for example, a small area of denudation is unlikely to be detected in microscopic examination of cross sections o f the vessel wall. Scanning electron microscopy (SEM), however, provides a tool for a rapid and t h o r o u g h examination o f large areas of vascular surfaces in great detail. Many square meters o f the endothelial surfaces of rabbits, rats, monkeys, and o t h e r species have been e x a m i n e d by SEM. These studies, in both normal and hyperlipemic animals, have shown a continuous layer of endothelial cells covering the surface, with no evidence of denudation until after lesion formation. 49-se This indicates that denudation either did not occur in the unmanipulated animal or involves areas that are too small to be detected by SEM. Possible explanations for this lack o f evidence for discontinuities come f r o m estimates o f the amount o f d e n u d e d surface likely to be present in normal animals or animals with moderate increases in turnover. Studies in the rat show that endothelium can recover an area one cell wide within about three hours. 5s T u r n o v e r studies in the same species imply a rate o f cell area loss o f approximately 1 x 10 -3 per day. 54,55 These values may be combined to estimate

VASCULARSMOOTH MUSCLE PROLIFERATION[Schwartz & Reid,/}

the total average area of denudation present at one time: E=Axrxt E = 0.125 Ixm~/cell where E = d e n u d e d area (I-tm~/ceU), A = area of each cell (103 la.m~), r = rate of t u r n o v e r (10 -3 days-i), t = time required to replace each cell (3 hours). T h e assumptions used in this calculation should result in a maximal estimate of the amount of denudation present during normal turnover. Nonetheless, 0.125 ~m 2 is a very small area that might be difficult to detect at the usual level of resolution available by SEM. This value, of course, could be much greater if turnover were increased. T h e r e is substantial evidence for increases in the turnover of endothelial cells in response to risk factors normally associated with the development of atherosclerosis. In experimental hypercholestero!emia, there is an increased thymidine labeling of the endothelihm in atherosclerotic lesions, 56 and this increase in DNA synthesis may precede the development of the lesions. 57 While these studies demonstrate a possible role for hypercholesterolemic endothelial injury in the formation of atherosclerotic lesions, endothelial denudation has been demonstrated in prelesion.stages, and is rare even after lesions are established? L56.Ss,~ Hypertension also causes an increased frequency of cell replication in the rat aortic endothelium. 6~ This is associated with a marked change in the structure of the aortic endothelium, including increased amounts of actin filaments,sl but there is no evidence for denudation of large vessels in hypertension. Whatever the mechanism leading to cell i0ss, there is an apparent discrepancy between the lack of evidence for denudation despite evidence for increased turnover. Studies of endotoxemia appear to resolve this paradox. Endotoxin treatment leads to an elevation o f the thymidine index of aortic endothelial cells. 69 Again, there have been only episodic r e p o r t s o f d e n u d a t i o n . S t u d i e s by R e i d y a n d Schwartz 6s in the endotoxin-treated rat showed that turnover increases without formation of d e n u d e d areas. The presence of cells loosely attached to the monolayer implied that this lack of denudation is a result of the coordinated undermining of detaching cells by adjacent viable endothelial cells. At least in this case, we appear to have a form of nondenuding desquamation. Before leaving the subject of endothelial denudation, the possibility that defects in endothelial integrity may occur in certain situations that are not readily detectable by scanning electron microscopy should be noted. T h e basis for this statement comes from recent studies where we found that smooth muscle cells can masquerade as endothelium; at least in terms o f their nonthrombogenicity and general morphology. Thus far we know this occurs when an artery has been d e n u d e d with a balloon catheter. Under these conditions, smooth muscle cells migrate 243

into the intinm and form a pseudoendothelium that, somewhat surprisingly, is not always overgrown by r e g e n e r a t i n g e n d o t h e l i u m . T h e s e smooth muscle cells are nonthrombogenic and, without careful examination with specific cell markers, can be readily mistaken for endotheliumP 4 Thus, it may he possible that previous studies have reported the presence of an intact endothelium when in fact the vessels may be lined by smooth muscle cells. In summary, we do not have evidence for spontaneous denudation, although we know that endothelial cells undergo spontaneous loss and replacement. I f denudation does occur in large arteries of hypertensive or atherosclerotic animals, it is either a rare event or, in the latter case, a late event. This implies that any causal role in atherosclerosis is likely to be related to the progression of already existing lesions. T h e endothelium is very delicate, and loss of the normal barrier functions should greatly accelerate lesion progression. As for hypertension, it seems likely that high blood pressure would accelerate lipid entry once endothelial continuity is lost. However, we have no evidence that hypertension accelerates the smooth muscle proliferation characteristic of atherosclerosis by causing endothelial injury in large vessels. CAUSES OF SMOOTH MUSCLE CELL PROLIFERATION If loss o f endothelial integrity is not an adequate explanation for control of smooth muscle proliferation, we need to consider other possible causes. Russell Ross and his colleagues 8 described the role of platelets and PDGF as a mitogen of smooth muscle cells in culture. They proposed that smooth muscle proliferation began with endothelial denudation followed by thrombosis and release of PDGF. In this view, proliferation is largely a reactive process. At the same time, Benditt proposed a totally different mechanism. He demonstrated that smooth m u s c l e p r o l i f e r a t i o n in h u m a n a t h e r o s c l e r o t i c plaques was a focal phenomenon apparently beginning in one or, at most, a few small muscle cells. 4 On the basis of the resemblance of the atherosclerotic lesion to smooth muscle tumors in the uterus, Benditt proposed that the proliferation was a monoclonal g r o w t h o r i g i n a t i n g f r o m some rfire m u t a g e n i c event. 65 He supported this hypothesis by showing that atherosclerotic lesions often contain one of two allogenic forms of glucose-6-phosphate dehydrogenase (G-6-PD). This was a striking finding because G-6-PD is sex linked, and in heterozygotic individuals, each cell expresses only one allele. Tiros, expression of a single allele in a lesion implies origin from one or at most a few cells. T h e essential difference from Ross's hypothesis may be that Benditt's hypothesis localizes the cause of smooth muscle proliferation to the smooth muscle cell itself, or at least intrinsic changes in the vessel wail, and suggests that

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the lesion begins in a single cell or no more than a small number of cells. RESPONSE TO INJURY AS A CAUSE OF SMOOTH MUSCLE PROLIFERATION

Ross's view has not been augmented by the discovery of several other potential sources for stimuli to smooth muscle proliferation. Besides the platelet, PDGF can be secreted by several cells including the macrophages, endothelial cells, and smooth muscle cells.6S-69 The possible importance of macrophage PDGF is obvious. At least in experimental animals, macrophage accumulation is a critical, early event in lesion formation, occurring prior to any evidence of endothelial denudation. 7~ It certainly occurs prior to loss o f endothelial continuity. M a c r o p h a g e s are prominent also in the uncomplicated lesion. It is possible that smooth muscle proliferation and lipid accumulation are both dependent on the same e v e n t ~ macrophage invasion of the wall. This raises a new issue. Do inflammation events precede lipid accumulation and cell proliferation? There is no answer as yet. This set of observations suggests what might be called the macrophage hypothesis. Like the platelet hypothesis, this theory proposes that smooth muscle proliferation occurs secondarily to some form of endothelial i n j u r y ~ i n this case, a loss of the normal functional ability o f the endothelium to exclude leukocytes from the vessel wall. Similarly, we could propose hypotheses linked to any of the other potential sources of mitogen endogenous to the vessel wall itself. ENDOGENOUS CONTROLS OF SMOOTH MUSCLE PROLIFERATION b

T h e discovery that the growth factor found in platelets is made also by endothelium and smooth muscle cells provides a possible explanation of Benditt's evidence that atherosclerotic lesions arise as an intrinsic change in the proliferative properties of the vessel wall cells. Endothelial cells secrete mitogenic peptides. 66 As already noted, we know that part, but not all, of this activity is attributable to PDGF. s7 More important to a possible endothelial hypothesis is the recent evidence that this ability of endothelial cells is modulated. PDGF is a translation production of the cis oncogene. In vitro, endothelial cells transcribe this gene at a high rate, but messenger RNA levels of cells isolated from endothelium in vivo are very low. 72 This reveals two things. The expression of cis in vitro is consistent with the idea that the PDGF-Iike material produced by endothelial cells is the same as the protein PDGF identified from the platelets and shown by sequence to be homologous to cis. The lack of expression in vivo suggests that expression in vitro is either an artifact or analogous to a response to injury 244

in vivo. Support for the latter hypothesis comes from a recent report of Jaye et al. 73 These authors have previously noted that endothelial cells can be induced in vitro to "differentiate" from flat sheets into tubes resembling simple blood vessels. They now find that expression o f cis is greatly reduced when endothelial cells enter the tube form. 7s While one could propose as an endothelial hypothesis that altered endothelium produces smooth muscle proliferation, this cannot explain proliferation in the absence o f endothelium. Furthermore, neither reactive mechanisms nor the endothelial hypothesis offers a simple explanation of Benditt's observation o f monoclonality of lesions. To what extent is the smooth muscle proliferation of atherosclerosis an endogenous process? Could a mutagenic event of the sort proposed by Benditt activate the PDGF gene in vascular smooth muscle cells? That is, to what extent does smooth muscle proliferation result from changes in the normal ways vessel wall cells relate to one another? Some clues come from a detailed analysis of cell proliferation in the balloon denudation model. As already noted, these experiments formed much of the original basis for tile hypothesis that thrombosis was the cause of smooth muscle proliferation in atherosclerosis. Total denudation via the balloon is followed immediately by platelet adherence. This is followed by medial smooth muscle DNA synthesis, beginning about two or three days after manipulation. 74-77 While the stimulated smooth muscle cells continue to replicate for a number of weeks, recruitment of cells into the growth fraction is largely completed within three days. 4s Heparin infusion inhibits the proliferative response but only if heparin is given within the first two days. 7s This suggests that heparin acts by preventing some, as yet undefined, early event that is critical to this response. Further support for this hypothesis comes from studies of the effects of heparin fractions on smooth muscle proliferation. Castellot et al. 79 and Fritze et al. s~ have reported that certain fractions o f heparan sulfate can inhibit growth of smooth muscle in culture, raising the interesting possibility that the initial event "committing" smooth muscle cells to growth may be released from inhibition rather than stimulation of growth. An understanding of the relationship of endothelial denudation to smooth muscle proliferation requires that we expand on the discussion o f the temporal sequence of events following balloon denudation. A d h e r e n c e o f platelets to the surface with release to platelet-granule products into the vessel wall occurs within minutes, sl,s2 Few new platelets adhere after the first few minutes, and the surface returns to low levels of thrombogenicity by 24 hours, s3 If platelet release is important, this period may be critical to the later sequence of events. Other blood cells are also found adherent to the surface of ballooned vessels, including neutrophils and mononuclear cells. While these cells may play a role in some species, they are not found in the rat and therefore

VASCULARSMOOTH MUSCLE PROLIFERATION[Schwartz & Reid,/)

are not, at least in this species, required for stimulation of smooth muscle proliferation. 33 When the carotid artery of a rat is ballooned, smooth muscle cells begin to enter the cell cycle between one and three days after the injury. Daily labeling-rate data show a continuation of cell replication over a period o f one month. 4s Similarly, DNA content of the intima" continues to increase over this interval. In contrast, the cumulative growth fraction, that is, the proportion of the original population that goes from a nonreplicating state into the cell cycle, rises rapidly over the interval between one and three days as expected but then remains essentially constant after day 7. In other words, those cells that are going to contribute to lesion growth become'committed to replicate very early after injury. This committed population of about 30 per cent of the cells in a carotid artery is then responsible for the bulk o f the growth of the ballooned vessel. Interestingly, not all of the cells that enter the intima replicate. About one of nine intimal smooth muscle cells is not labeled, even with continuous labeling. Based on an estimate of the number of divisions in each cell, we estimate that 50 per cent of the cells that cross the internal elastic lamina never divide. 4s It is reasonable to speculate that the commitment of cells to enter the growth fraction may correlate with migration o f smooth muscle cells from the media into the wounded intima. As shown by morp h o l o g i c a n d by c y t o k i n e t i c studies, 48,70,71,74,84 smooth muscle cells migrate across the vessel wall. As m e a s u r e d by 3H-thymidine autoradiography, the first arrival o f cells in the intima does not occur until after cell replication has begun. 7~ This is important because it rules out the possibility that the initial entry of cells into the cell cycle, as described in the previous paragraph, requires relocation of smooth muscle cells into the intima. However, the possibility remains that continuation of replication, i.e., division of committed cells, may depend on this translocation. If this is true, then one might speculate on the possibility that migration is critical to the maintenance of a proliferative state in the ballooned wail. As already discussed, the thrombotic response to denudation is extremely transitory. By 48 hours, the number of platelets adhering to the denuded vessel is less than half the number observed acutely after injury. s5 This low level of 51Cr uptake persists for several months, perhaps related to the persistence, as m e n t i o n e d earlier, o f denudation. If this initial platelet response is critical to proliferation, then we would expect smooth muscle proliferation even with a n a r r o w e n d o t h e l i a l wound. A critical role o f platelets in the response to the balloon is supported by the observation that antiplatelet serum inhibits proliferation in the injury model, s6-s9 Since, however, proliferation does not occur with milder forms o f endothelial abrasion, the acute thrombotic response is not a sufficient stimulus. 47 While persistence of small amounts of plateletreleased materials could be important for continued 245

replication of smooth muscle, these experiments suggest that some other event in the first three days initially commits the smooth muscle cell to replication. The acute events induced by injury of platelet release or other factors could induce a persistent change in tile proliferative potential o f the smooth muscle cells. For example, migration is an early event. Migration of dividing cells into the intima could be required for subsequent proliferation if cells in the intima are released from density-dependent controls present in tile densely populated tunica media. In turn, this hypothesis would require either some sustained change in the smooth muscle cells in the intima or in their environment that would permit these cells to continue growing long after the initial thrombotic response has been ended. Other hypotheses need to be considered. We have shown recently that dead smooth muscle cells release a cytoplasmic factor that is mitogenic for other smooth muscle cells. 9~ In our hands, injury produces trauma sufficient to cause total endothelial denudation with the balloon catheter, which is always associated with smooth muscle cell death. 9~ It is interesting to note that Imai and his collaborators 92 showed that oxidized dietary cholesterol was a more effective atherogen than unoxidized cholesterol and that this difference could be correlated with occnrrence o f smooth muscle cell death in animals receiving the oxidized material. Putting these observations together, one might posit that mitogens released from dying cells are required for stimulation of smooth muscle replication in vivo. Platelet factors released as part o f the injury response might have several effects related to the important question of localization of cells to the intima. PDGF is not only mitogenic, it is chemotactic for smooth muscle cells. 93 Platelets also contain TGF-[3, a p o l y p e p t i d e that is not mitogenic but, when presented with a mitogen, promotes growth of normal cells in soft agar. 94 TGF-[3 could be required for growth o f smooth muscle cells outside the media. Smooth muscle cell movement into the intima and growth in that location could require a chemotactic stimulus (e.g., PDGF), a stimulus to growth without anchorage, and breakdown of the extracellular matrix, perhaps resulting from a combination of release o f proteases from necrotic cells and mechanical disruption by distension. Finally, an alternative possibility for the maintenance of growth is offered by recent studies in our laboratories. We attempted to d e t e r m i n e the cell cycle status of quiescent cultures of smooth muscle cells o f the n e w b o r n rat by placing these cells in plasma-deficient serum. T o our surprise, growth of these cells could not be arrested under these conditions. When the conditioned medium was examined for mitogenicity, the culture medium was able to stimulate 3T3 cell and smooth muscle cell replication. T h e mitogen responsible for this activity is inactivated by anti-PDGF antibody, and the conditioned medium competes for the PDGF receptor. Thus, it

HUMAN PATHOLOGY

Volume 18, No. 3 (March '1987]

appears likely that fetal smooth muscle cells, at least in one species, can make this important m!togen. 69 F u r t h e r m o r e , t h e r e is also evidence that adult smooth muscle cells can be induced to produce similar material. Cultured rat arterial smooth muscle cells from adult animals produce low or absent levels of PDGF-Iike material. However, when cells were derived from a ballooned vessel two weeks after injury, these cells also produced PDGF-Iike material. 95 It is important to note that these observations were made only on cells placed in culture; we have no direct evidence for comparable levels of production in vivo. Nonetheless, the appearance of activity in cells derived from the balloon-injured vessel implies that some dramatic change has occurred in the capabilities of the smooth muscle cells making up the injured vessel wall. It is intriguing to consider the possibility that this change could be responsible for both the commitment to replication and the maintenance o f replication during the days and weeks after injury. We would like to offer a speculation. Commitment of smooth muscle cells in the ballooned vessel to continued replication and the behavior of cultured cells derived from these ballooned intima could be accounted for in one of two ways. First, the balloon might activate s o m e p r o p e r t y o f all vessel wall smooth muscle cells so that these cells maintain or develop the ability to produce PDGF. Alternatively, the balloon could selectively activate a subpopulation of cells that retain the properties o f the i m m a t u r e smooth muscle ceils found in the pup aorta. Tiffs latter idea is attractive for several reasons. A small p o p u l a t i o n o f residual i m m a t u r e "blast" cells is known to exist in skeletal muscle and to play a role in regeneration of this muscle. 96,97 If, as a response to the balloon, a few widely scattered immature smooth muscle blast cells were stimulated to nfigrate into the intima, the cells could be released from controls of proliferation imposed by neighboring more mature cells. In the intima, these cells might not only proliferate but also might p r o d u c e mitogens for other cells. This form of selective proliferation of a subset of cells is consistent with our kinetic evidence that only 50 per cent o f cells crossing the intima in response to the balloon go on to proliferate. More importantly, selection among a smooth muscle population to a subpopulation already having a proliferative advantage could account for both the monoclonal phenotype of chronic human atherosclerotic lesions 65 and suggests from others that monoclonality arises gradually as the human lesion evolves. 12,98 Obviously, the idea that stem cells exist at all is highly speculative. The idea that such a phenomenon might be imp o r t a n t in atherosclerosis d e p e n d s on k n o w i n g whether the proposed blast ceils exist in man and if they do, what proportion o f ceils forming this lesion has these properties. REFERENCES 1. Robbins SL, Cotran RS: Pathologic Basis of Disease, ed 2. Philadelphia, WB Saunders, 1979 2. Velican C, Velican D: The precursors of coronary atherosclerotic plaques in subjects up to 40 )ears old. Atherosclerosis 37:33, 1980

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