Growth factors and the pathogenesis of atherosclerosis

Afherosclerms, 62 (1986) 185-199 Elsevier Scientific Publishers Ireland,

185 Ltd.

ATH 03838

Review

Growth Factors and the Pathogenesis

of Atherosclerosis

Jan Nilsson Department of Histology, Karolinska Insiitutet, and Department of Medicine, Danderyd Hospital, Stockholm (Sweden) (Received 19 February, 1986) (Revised, received 20 July, 1986) (Accepted 25 July, 1986)

Introduction Atherosclerosis is a disease characterized by a focal intimal thickening of medium and large-sized arteries. Its clinical manifestations include cerebral and myocardial infarction. Epidemiological studies have identified several factors associated with an increased incidence of coronary heart disease and cerebrovascular disease, such as hypercholesterolemia, hypertension, smoking and diabetes, but it remains to be clarified if these factors are mainly atherogenic or thrombogenic. Some further information have been provided by recent angiographic studies that have demonstrated an association between high levels of low density lipoprotein cholesterol and coronary atherosclerosis [l-3]. However, since the early stages of lesion formation are characterized by intimal cell proliferation as well as lipid infiltration, it is not possible to explain the etiology of atherosclerosis simply in terms of cholesterol deposition in the arterial wall. On the basis of the finding that platelets contain a potent mitogen for smooth muscle cells (SMC) Ross and Glomset suggested that atherosclerotic lesions develop in response to endothelial denudation and subsequent platelet adherence, and release of growth factors [4]. Lately this hypothesis has been questioned since it has been difficult to demonstrate a clear association between endothelial denudation and development of atherosclerosis in vivo [5]. An alternative concept has developed during recent years as it has become Correspondence to: Jan Nilsson, Department of Histology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden. 0021-9150/86/$03.50

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clear that mitogens can be produced also by cells present in the arterial wall [6-91. These findings raise the possibility that stimulation of SMC replication may be initiated independently of platelet aggregation and release of platelet mitogens and instead occur as a result of an endogenous process in the arterial wall. This review focuses on the role of SMC proliferation and phenotypic alteration in development and progression of atherosclerosis. It further discusses different hypothesis concerning initiation and regulation of intimal SMC growth. Histopathological Observations ment of Atherosclerotic Lesions

on the Develop-

In animals, experimentally induced atherosclerosis is achieved either by a dietary-induced hypercholesterolemia or by producing an endothelial denudation and arterial damage by mechanical or toxic injury. In view of the different nature of these two methods it is not surprising that the developing lesions morphologically appear somewhat different. After a few days on hypercholesterolemic diet monocytes can be found adhering to the endothelial surface [lo-131 and signs of increased endothelial turnover are noted [14]. Within a month areas with intimal edema are observed and monocytes have penetrated the endothelial barrier and accumulated as resident macrophages. Accumulation of lipid-filled intimal macrophages (foam cells) protruding into the vessel lumen, represent the earliest type of atherosclerotic lesion, the fatty streak. At a later stage proliferating SMC give rise to fibrous plaques at the same anatomical Ltd.

186

Fig. 1. Different stages in development of atherosclerotic lesions. A: The normal, unaffected arterial wall. B: The intima is infiltrated by monocytes that ingest lipids and turn into foam cells. Clusters of foam cells give rise to a fatty streak. C: Modulation of smooth muscle phenotype from contractile to synthetic. The smooth muscle cells penetrate through the internal elastic lamina and migrate into the intima. D: Proliferation of smooth muscle cells in the intima. Endothehal denudation and platelet adherence may occur at this stage. E: Formation of an advanced irreversible atherosclerotic lesion with a dense cap of fibrous tissue and a central core of lipids and necrotic tissue.

187 sites [ll-131. In spite of extensive research the evidence for conversion of fatty streaks into fibrous plaques remains circumstantial [15] and it is interesting to note that fatty streaks in swine appear to begin at sites of pre-existing intimal SMC masses [16]. In the majority of the studies in which dietary hypercholesterolemia is used to induce atherosclerosis early lesions develop without any signs of endothelial injury or platelet engagement [11,16-211. However, in a study on nonhuman primates Faggiotto and Ross [13] found signs of endothelial denudation after many month of high-cholesterol diet. The principal stages of lesion formation are outlined in Fig. 1. Mechanical removal of the arterial endothelium causes a rapid adherence of platelets to the exposed subendothelial tissue [22-251. If the wound is deep enough to affect the media, SMC start to migrate into the intima where they synthesize DNA and divide. In a couple of months several layers of SMC surrounded by collagen fib&, elastic fibres and proteoglycans give rise to a fibrous plaque [22,23,26-301. However, a superficial injury restricted to the endothelium does not give rise to SMC proliferation in spite of abundant platelet adherence [ 311. Although there are many similarities, atherosclerotic lesions in humans are not necessarily of the same origin and nature as those seen in experimental animals. In man fatty streaks and areas of edema are frequently observed in the coronary arteries during the first 3 decades of life, whereas after the age of 30 these lesions are gradually replaced by fibrous plaques and other advanced lesions [32-351. The majority of fine structural studies on human atherosclerotic lesions have been performed on material obtained at autopsy. However, the poor preservation of the material has made a positive identification of the cellular components difficult. In a recent study Ross et al. [36] made a careful characterization of lesions obtained from occluded femoral arteries during by pass surgery. These lesions consisted of SMC and occasional macrophages, covered by a dense cap of connective tissue. Although these lesions were large enough to occlude the vessels, signs of degenerative changes and lipid inclusions were rare. Jonason and coworkers [37] used an immunological technique to analyse the cellular composition

of plaques removed from the internal carotid artery during surgery. SMC were found to dominate in the fibrous cap, which also contained many T cells, whereas the macrophage was the most frequent cell type in the central lipid core. SMC Proliferation and Lesion Formation Current concepts of the etiology of atherosclerosis have focused on the role of SMC proliferation. It has been suggested that lesions develop in response to endothelial injury [4]. According to this model platelets adhere to exposed subendothelial tissue, aggregate and release platelet-derived growth factor (PDGF) and possibly other platelet mitogens into the vessel wall. In the intima PDGF stimulates recruitment, proliferation and synthesis of the connective tissue matrix of SMC. If the intimal injury is continuous or repeated, lesion formation may progress. As opposed to the original ‘response to injury’ hypothesis several investigators have proposed that intimal SMC replication occurs as a result of changes intrinsic to the arterial wall rather than in response to exogenous mitogens. In 1973, Benditt and Benditt [38] reported evidence of a monoclonal origin of human atherosclerotic lesions. This conclusion was based on the finding that cells from human atherosclerotic lesions only contained 1 of the 2 isoenzymes of glucose-6-phosphate dehydrogenase (G-6-PD), whereas cells from the surrounding arterial wall expressed both types of the enzyme. This enzyme is coded on the X chromosome and is expressed randomly in its A or B type in heterozygous individuals. One possible explanation of this finding is that the lesions have originated from proliferation of a single stem cell. The authors suggested that the neoplastic growth characteristics of the stem cell were induced by chemical mutagens or viruses. G-6-PD monotypism in human atherosclerotic lesions has also been demonstrated by Pearson et al. [39] and by Thomas et al. [40]. However, the latter found G-6-PD monotypism in only 30% of the lesions investigated. Of the monotypic lesions investigated by the group in Thomas laboratory more than 80% were of the same type as that predominant in the surrounding arterial wall. On the basis of these findings it was suggested that G-6-PD

188 monotypism in human lesions did not occur as a result of monoclonal proliferation but due to a ‘phenotype-selective advantage’ [16]. An alternative model have been proposed by Martin and Sprague [41], who suggested that the normal SMC population in the arterial wall is maintained by proliferation of a subpopulation of stem cells. Replicating stem cells differentiate into SMC, which subsequently produce growth-inhibitory ‘chalones’ that inhibit further stem cell proliferation by a negative-feedback mechanism. In old age the SMC ‘in the arterial wall become senescent and produce less growth-inhibitory ‘chalones’. In the densely populated media the concentration of the ‘chalones’ would still be high enough to inhibit stem cell proliferation whereas the concentration in the intima would be too low to inhibit clonal proliferation of stem cells. A similar model has also been proposed by Schwartz et al. [42] who suggested that the arterial media contain smooth muscle ‘blast’ cells that are growth-inhibited by the influence of neighboring more mature cells. If the ‘blast’ cells are stimulated to migrate into the intima, this control mechanism is lost and the cells would continue to proliferate and thus give rise to a monoclonal lesion. More recently Campbell and Campbell [43] have focused on the importance of intrinsic changes of SMC phenotype in development of atherosclerotic lesions. They suggest that SMC in intimal lesions express a phenotype that make them more responsive to mitogenic stimulation. Endothelial Integrity and Atherogenesis

Much of the early interest in the role of endothelial integrity in atherogenesis was evoked by the finding that mechanical deendothelialization by a balloon catheter caused SMC proliferation and intimal thickening [44,45]. As suggested by Ross [4] it seemed reasonable to assume that the intimal proliferation of SMC occurred in response to PDGF released from aggregation platelets. However, in contrast to this model, several investigators have demonstrated development of early stages of atherosclerotic lesions without any signs of endothelial denudation [11,16-21,461. It is possible that denuded areas exist but have been over-

looked because they have been too small to discover by scanning electron microscopy. On the other hand, a l-2 cell wide endothelial denudation closes within 8 hours and does not give rise to intimal SMC proliferation [47,48]. Reidy and Silver [31] also reported that no SMC replication occurred after a 90-120 cell wide removal of the endothelium in spite of abundant platelet adherence. Moreover, most platelet adherence and release is restricted to the first 2-3 h after endothelial removal with a balloon catheter [25,49] and platelet factors can only be detected in the vessel wall during the first 4 h after denudation [50]. SMC replication, however, continues for several weeks 1511. It has been proposed that the discrepancy in SMC replication reflects a difference in the magnitude of the inflicted injury and that SMC proliferation only occurs when the injury affects the media [26]. Further evidence supporting this concept have recently been presented by Walker et al. [52] who found intimal thickening after deendothelialization with a rigid steel wire, whereas a similar endothelial denudation caused by a flexible nylon filament did not result in formation of an intimal plaque. Nevertheless it is possible that endothelial dysfunction other than actual denudation is involved in atherogenesis. Hypertension and hyperlipidemia have been found to be associated with an increase in endothelial cell turnover [14,51,53,54] and the rate of endothelial replication appear to be highest at branching points known to demonstrate a high incidence of atherosclerotic lesions [55]. In hypercholesterolemic animals of most species early lesions are associated with increased endothelial permeability and adherence and penetration of blood-borne monocytes [lo-13,561. In contrast, Thomas et al. [57] found engagement of monocytes only in advanced lesions of hyperlipemic swine. It is in this concept also interesting to, note that young patients with coronary atherosclerosis have increased levels of PDGF-like mitogenic activity in plasma [58] that may penetrate into the intima in areas of increased permeability. Intimal infiltration of macrophages may be an early key event in atherogenesis and identification of the factors responsible for recruitment of monocytes remains one of the major unsolved problems of atherosclerosis research. A

189 factor with chemotactic effect on monocytes has been isolated from aortic tissue underlying areas with increased endothelial permeability [59] and from cultured endothelial cells exposed to angiotensin [60]. The inflammatory/immune mediator interleukin 1 alters the surface properties of endothelial cells in such a way that monocyte adhesion is induced [61] and monocytes are also attracted to IgG that accumulates in injured endothelial cells [62]. In contrast, low density lipoproteins modified by endothelial cells inhibit the migration of macrophages and may thus trap them in the lesions

[631. Modulation of Smooth Muscle Cell Phenotype Depending ment arterial

on the requirements of the environSMC express different phenotypes.

In young developing arteries the SMC have the ability to proliferate and produce large amounts of collagen, elastin and proteoglycans to the surrounding matrix. In the adult artery their main function is to regulate wall tension, but they may also modulate back into ‘synthetic’ phenotype and participate in tissue repair. SMC engaged in the formation of atherosclerotic lesions also appear to regain the phenotypic characteristics of SMC found in young developing arteries [64,65]. This modulation enables the cells to proliferate and to produce extracellular matrix components, which represents two critical events in lesion formation. The SMC of the adult artery that have a cytoplasm filled with myofilaments and that contract in response to vasoconstrictors and the SMC of embryonic arteries that secrete matrix components

Fig. 2. A: Smooth muscle cell fixed directly after isolation The cytoplasm is dominated by myofilament bundles (F) and associated dense bodies (DB). Bar 0.5 pm, X 24000. B: Smooth muscle cell fixed after two days in medium supplemented with 10% serum. Most of the cytoplasm is still filled with filaments but the rough endoplasmic reticulum (RER) has started to grow out from the nuclear envelope. Bar 0.25 pm, X49000. C: Smooth muscle cell fixed after 4 days in serum-containing medium. The filaments have now disappeared almost completely and are replaced by RER and a prominent Golgi complex (GC). Bar 0.25 pm, x 54000. Mitochondria (M), nucleus (N) and lysosome (L).

190 represent the two extremes of the phenotypes expressed by arterial SMC. Based on their functional and morphological characteristics they have been referred to as being in ‘contractile’ or ‘synthetic’ phenotype [66]. Modulation from ‘contractile’ to ‘synthetic’ phenotype can be studied by using primary cultures of enzymatically isolated arterial SMC. Early in culture the cells are rounded and their cytoplasm is dominated by myofilaments and associated dense bodies (Fig. 2A). Transition into ‘synthetic’ phenotype starts within 2-6 days (depending on the species studied) with an outgrowth of rough endoplasmic reticulum (RER) from the nuclear envelope (Fig. 2B). Within a few days all cells have completed transition into ‘synthetic’ phenotype and filaments are now found mainly in the cell periphery, while the cytoplasm is dominated by an extensive RER and a prominent Golgi complex [67-701 (Fig. 2C). After completing the modulation process the SMC proliferate rapidly until confluence is reached. Chamley-Campbell and Campbell have reported that SMC that reach confluence within five doublings spontaneously revert back into ‘contractile’ phenotype, while cells that have undergone more than five doublings irreversibly remain in ‘synthetic’ phenotype [71], suggesting an irreversibel change in gene expression. Our knowledge about signals initiating modulation into ‘synthetic’ phenotype is still very limited. In the arterial wall it appears to occur in association with tissue damage and/or infiltration of macrophages. In culture modulation into ‘synthetic’ phenotype is inhibited if SMC are grown in the presence of confluent layers of endothelial cells or contractile SMC [71], whereas PDGF and PGE, have been found to enhance the progress rate of the modulation process [68,72]. The modulation process is also inhibited by heparin [71] and it has been suggested that the endothelial-derived inhibitory factor is a heparinlike molecule released from the surface of endothelial cells by a platelet endoglycosidase heparitinase [73]. Platelet-derived Growth Factor and Autocrine Stimulation of Smooth Muscle Cell Growth Platelet-derived growth factor (PDGF) is a major serum mitogen for SMC and other connec-

tive tissue cells. PDGF is a 30-kD basic protein consisting of two disulphide-linked polypeptide chains (A and B). It is stored in the a-granulae of platelets and released during blood clotting. Since the cellbiological effects of PDGF also includes chemotaxis and stimulation of secretion of matrix components, it seems reasonable to assume that PDGF functions as a local wound hormone. The early response to activation of the PDGF receptor includes a tyrosine-specific autophosphorylation of the receptor, an increase in intracellular Ca*+ concentration, an extrusion of protons via the amiloride-sensitive Na+/H+ exchange carrier, and an internalization of the ligand-receptor complex. Stimulation with PDGF also induces RNA synthesis and production of a number of specific proteins, among them the proteins coded by the cellular oncogenes fos and myc (for a recent review on PDGF see [74]). The identification of the viral oncogenes (v-one) and their cellular homologues, the protooncogenes (c-one), has greatly increased our understanding of the regulation of normal cell proliferation. These genes appear to code cellular proteins that play important roles in the regulation of cell proliferation, and quantitative and qualitative changes in the expression of these genes have been implicated in neoplastic transformation (for review see [75]). The amino acid sequence of the B chain of PDGF is almost identical to that of the transforming protein of simian sarcoma virus (SSV) p28sis, encoded by the viral oncogene v-sis [76,77] and entirely homologous to the predicted product of its normal cellular homologue, c-sis [78]. SSVtransformed cells produce a mitogen that resembles PDGF immunologically and functionally [79,80] and may thus stimulate their own growth in an autocrine manner by activation by the PDGF receptor. Similarly, expression of c-sis and production of a PDGF-like protein have been demonstrated in a number of sarcoma and glioma cell lines [81,82]. There is also evidence that endogenous PDGFlike proteins stimulate growth of SMC in an autocrine manner. The ductus deferens smooth muscle tumour cell line DDT, MF-2 expresses c-sis and produces a protein which is immunologically identical to PDGF. Glucocorticoids inhibit both c-sis expression and cell growth, in these cells. This

191 inhibition is overcome by addition of exogenous PDGF [83,84]. Seifert et al. [6] recently reported that secondary cultures of SMC from newborn but not from adult rats produce a PDGF-like protein, suggesting that autocrine stimulation of arterial growth may be of importance during neonatal development. As discussed above, adult arterial SMC in primary culture modulate from ‘contractile’ to ‘synthetic’ phenotype. This transition is followed by a phase of very rapid growth which is partly independent of exogenous mitogens [68] and accompanied by an endogenous production of a PDGF-like protein [7]. The ability to grow in serum-free medium ceases after 8-12 days in primary culture as the cells reach confluence, but reappears if a wound is produced in the culture by scraping away the cell in a 2-3 mm wide corridor [7]. Somewhat surprisingly it has not been possible to identify sis (B chain) transcripts in cultured arterial SMC. Instead these cells have been found to express the gene for the A chain and the PDGF-like mitogen produced by arterial SMC is probably a A chain homodimer [85]. In accordance with these findings Walker et al. [86] have reported that SMC isolated from arterial intima two weeks after a balloon catheter-induced injury produced ten times as much PDGF-like material as SMC isolated from the media of uninjured arteries. Taken together these results suggest that SMC which as a result of some type of injury have modulated into ‘synthetic’ phenotype and migrated into the intima regain the functional characteristics of SMC in young developing arteries including expression of the gene coding the A chain of PDGF, secretion of a PDGF-like mitogen and an autocrine stimulation of cell growth. At least in cell culture the ability to stimulate growth via an autocrine loop is restricted to a limited period after the modulation into synthetic phenotype [7]. Further support in favour of this concept comes from the studies of Grunwald and Haudenschild [87] who found that SMC isolated from intimal explants from balloon catheter-injured arteries proliferated at a higher rate than SMC from uninjured arteries, and that the growth of SMC from injured arteries became temporarily serum-independent. No studies have yet been designed to investigate if SMC engaged in spontaneous lesion formation express PDGF-like mitogens, but sever-

al reports have demonstrated that SMC isolated from early lesions both of animals [88,89] and humans [90] proliferate at a higher rate than SMC isolated from more advanced lesions or from the surrounding media. Other Growth-regulating Wall

Factors in the Arterial

It seems unlikely that regulation of cell replication in vivo is the result of a single stimulatory or inhibitory signal. A large number of diverse substances have been found to affect cell replication in vitro. Unfortunately we know very little about how these substances interact in their normal environment. The recent use of different coculture systems and in situ hybridization technique may help to clarify this important problem. In vivo growth of arterial SMC is probably influenced by factors released from endothelial cells and macrophages, by direct cell-cell contact and by factors released from neurons and the blood. The macrophage is the first cell type present in a developing atherosclerotic lesion of hypercholesterolemic animals. The occurrence of macrophages precedes the SMC proliferation by weeks or months. Macrophages release 12-r-hydroxy5,8,10,14-eicosatetraenoic acid (lZHETE), a lipoxygenase product of arachidonic acid that have been found to stimulate SMC migration in concentrations as low as lo-l5 g/ml [91]. Activated peritoneal macrophages but not circulating monocytes also produce a factor that stimulates growth of SMC and fibroblasts [92,93]. It was recently reported that at least part of the macrophage-derived mitogenic activity is due to production of a PDGF-like mitogen and that activated macrophages contain sis RNA transcripts [8]. Experimental evidence also indicates that macrophages stimulate proliferation of fibroblasts both in coculture [94] and in vivo [95]. Clearly macrophages play an important role in atherogenesis, but it remains to be elucidated whether they act as scavenger cells by removing intimal lipids or participate in lesion formation by stimulating SMC migration and proliferation. Endothelial denudation and replication may influence lesion progression, not only by allowing platelet adhesion and lipid penetration, but also

192 by production of substances promoting or inhibiting SMC proliferation. In animals with experimentally induced endothelial injury intimal SMC replication remains abundant in the regions last covered by regenerating endothelium [28]. Whether this is due to presence of growth-promoting substances originating from the blood or lack of growth-inhibitors derived from the endothelium is not known. In culture, endothelial cells have been found to produce both growth-promotors and growth-inhibitors. It is interesting to note that endothelial cells, with the exception of SMC and macrophages, are the only non-transformed cells known to express the gene for any of the PDGF chains [96,97] and to produce a PDGF-like mitogen [9]. In vitro, expression of c-sis is found in proliferating but not in quiescent endothelial cells [96,97]. Endothelial cells lack PDGF receptors and do not respond to stimulation with PDGF [98]. On the other hand, this mitogen may stimulate migration and proliferation of surrounding SMC. Endothelial cells have also been found to produce a mitogen unrelated to PDGF [99]. It remains to be determined whether the net effect of the growth-regulatory factors produced by the endothelium in vivo is stimulatory or inhibitory. However, recent co-culture experiments indicate that endothelial cells suppress the growth of co-existing SMC [loo]. A high concentration and rate of metabolism of adenosine and adenosine nucleotides is characteristic of vascular endothelium [loll. Adenosine [102], as well as prostaglandin E, [103], has been found to inhibit the mitogenic effect of PDGF on cultured SMC. There is a close relation between the inhibitory effect of these substances and their ability to raise the intracellular concentration of CAMP in SMC. Furthermore, endothelial cells release a heparin-like inhibitor of SMC growth [104]. Heparin inhibits SMC proliferation both in vitro [105] and in vivo after endothelial denudation [106]. The exact mechanism by which heparin inhibits cell proliferation is unknown but it has been shown that heparin binds to specific surface receptors on SMC and inhibits RNA as well as DNA synthesis [107,108].

Hypertension Hypertension is a well known risk factor for development of cardiovascular disease [109]. Somewhat surprisingly, recent clinical studies have demonstrated that antihypertensive treatment that effectively reduces ‘pressure-related’ complications, such as stroke and congestive heart failure, has much less effect on complications related to atherosclerosis, such as coronary artery disease [110]. One possible interpretation of these results is that the development of atherosclerotic lesions is not due to the increase in blood pressure in itself but to some other associated dysfunction. Early intimal changes in animals with experimentally induced hypertension or spontaneously hypertensive animals show close resemblance to those found in animals with experimentally induced hypercholesterolemia, including an increased endothelial turnover and permeability and an intimal infiltration of monocytes [46,111]. In acute hypertension a transient increase in SMC replication occurs during the first two weeks [112], whereas in animals with chronic hypertension SMC replication occurs at a later stage [113]. Arterial SMC from hypertensive animals are also characterized by a cellular hypertrophy and nuclear polyploidy [114]. In spontaneously hypertensive rats antihypertensive treatment prevented development of cellular hypertrophy and polyploidy but lacked effect on the increased rate of SMC proliferation [113]. There is an interesting relation between the pressor activity of vasoactive hormones and their influence on cell growth. Several hormones known to induce arterial contraction such as angiotensin [115], vasopressin [115] and norepinephrine [116] stimulate growth of cultured arterial SMC, whereas substances that induce vasodilation such as PGE, [103] and adenosine [102] inhibit growth of these cells. Furthermore, it has recently been demonstrated that PDGF is a potent vasoconstrictor [117]. Contraction of smooth muscle cells is medistores. ated by release of Ca * + from intracellular Angiotensin, vasopressin, norepinephrine and PDGF increase formation of inositol triphosphate (IP,) [118-1201, a substance recently recognized as the second messenger for mobilization of intracellular Ca*+ [120]. Formation of IP, and release of

193 Ca2+ from intracellular stores contribute to the early cellular response to growth factors [120]. Thus it is possible that an increased formation of IP, is a common characteristic of both hypertension and atherosclerosis. The finding that calcium antagonists are not only effective as antihypertensive drugs but also inhibit proliferation of SMC may prove to be of therapeutic value in the future [121,122]. Receptor-mediated dilation of smooth muscle is coupled to increase in the intracellular concentration of cyclic AMP. There is also a close relation between the ability of adenosine and prostaglandins to raise intracellular cyclic AMP and their ability to inhibit PDGF-induced initiation of DNA synthesis [101,102]. Exceptions to this general rule are the neuropeptides substance P and substance K, which both have vasodilatory properties and stimulate growth of arterial SMC [123]. However, the vasodilatory effect of these peptides is not mediated by a direct effect on the smooth muscle but by inducing release of an endothelial-derived relaxing factor [124]. It is not likely that vasoactive substances such as angiotensin and substance P are true growth factors in the same sense as PDGF. Instead they may act as general stimulators of cellular activity and enhance the mitogenic effect of suboptimal concentrations of other growth factors, such as PDGF. Hypertension has also been found to cause intrinsic changes in SMC response to growth factors. Haudenschild et al. [125] have demonstrated that SMC isolated from the aortic media of hypertensive rats have an increased rate of migration and replication as compared with SMC isolated from the aortic media of normotensive rats. This behaviour closely resembles that of SMC isolated after mechanical arterial injury [86]. Lipoproteins The association between high levels of low density lipoprotein (LDL) cholesterol and development of cardiovascular disease has been described in large scale population studies [126,127]. In fatty streaks and other early types of lesions cholesteryl esters accumulate in foam cells, whereas in more advanced stages of atherosclerotic plaques they are also frequently observed bound to the extracellular matrix. The extracellular lipids may

originate either from dead foam cells or from circulating lipoproteins. The rate of intimal lipid accumulation depends both on the function of the transport system for circulating lipoprotein-bound cholesterol and on the factors regulating cholesterol uptake in foam cells. A normal removal of circulating cholesterol in bile acids which are secreted into the intestine requires the presence of adequate levels of high-density lipoproteins (HDL) and hepatic apo B, E lipoprotein receptors (for reviews see [128,129]). In atherosclerotic lesions cholesterol primarily accumulates in foam cells. Since SMC have a limited ability to ingest LDL these cells appear largely to be derived from macrophages. Macrophages have very few binding sites for normal LDL but ingest large amounts of ‘charge-modified’ LDL via a high-affinity receptor, usually referred to as the ‘scavenger receptor’. In contrast to normal LDL receptors, the ‘scavenger’ is not downregulated in the presence of an excess of cholesterol, thus permitting uptake of large amounts of lipids and formation of foam cells [130]. The physiological role of the ‘scavenger receptor’ is still unknown. LDL which has been modified by incubation with endothelial cells [131] and oxidized LDL [132] bind to this receptor. Accordingly, macrophages have the ability to bind and ingest modified forms of intimal LDL and act as scavenger cells by removing cholesterol from the arterial wall. In the early stages of experimental atherosclerosis, induced by a hypercholesterolemic diet, lesions are characterized by macrophage infiltration and SMC proliferation rather than lipid deposition. Hence lipids seem to be involved also in the initiation of the proliferative response in the arterial wall. In this respect lipids may act by inducing an increased endothelial turnover and permeability that permits entry of monocytes from the circulation [14]. Lipoproteins may also enhance lesion progression by their ability to stimulate SMC proliferation [133] and platelet aggregation [134]. Development of Atherosclerotic Lesions A model explaining ment of atherosclerosis

the etiology and developmust take into account

194 several different aspects of the disease. - Which factors are responsible for the increased endothelial permeability and the intimal infiltration of monocytes seen in the early stages of the disease? - How is the transition of SMC into ‘synthetic’ phenotype initiated and which factors influence SMC replication in the arterial intima? - Which factors regulate accumulation and clearance of intra- and extracellular lipids in atherosclerotic lesions? Although we remain unable to present satisfactory answers to these questions our understanding of the complex cellular interactions involved in lesion formation have increased significantly over the last few years. It has become clear that PDGF released from aggregating platelets in connection with endothehal denudation is not a major initiating factor in atherogenesis. We know that cultured endothelial cells and macrophages produce a PDGF-like mitogen, but it remains to be clarified if this mitogen is produced in active concentrations in vivo. We also know that cultured SMC in connection with a change of phenotype produce a PDGF-like mitogen and temporarily proliferate independent of exogenous mitogens, but it is unclear if this is an important etiological mechanism in lesion formation. The ability of a single or subpopulation of SMC to stimulate growth by an autocrine mechanism could hypothetically give rise to monoclonal lesions or provide cells with a ‘phenotype-selective advantage’ [38-421. Based on the findings discussed in this review a modified version of the ‘response-to-injury’ hypothesis presented by Ross and coworkers [4] is proposed below. A long-term exposure of normal arteries (Fig. 1A) to irritating stimuli such as hypercholesterolemia, hypertension, anoxia or turbulent blood flow induces an inflammatory response in the arterial wall. This response includes increased endothelial replication and permeability, intimal edema and infiltration of macrophages. Resident macrophages ingest LDL-associated cholesterol modified by and transported into the intima by replicating endothelial cells. In the presence of an excess of intimal lipids macrophages turn into foam cells and give rise to fatty streaks (Fig. 1B). Intimal macrophages release several different

cycloxygenase and lipoxygenase products, among them lZHETE, and a PDGF-like protein. These substances, possibly in concurrence with a PDGF-like substance released from replicating endothelial cells, stimulate migration of SMC to the intima and their modulation into ‘synthetic’ phenotype (Fig. 1C). SMC in ‘synthetic’ phenotype regain the functional properties of SMC in young developing arteries, including production of a PDGF-like mitogen and autocrine stimulation of cell growth. The ability of autocrine growth stimulation is restricted to a limited period. However, additional growth-promoting factors are released from newly recruited SMC, macrophages and replicating endothelial cells. Free PDGF in plasma and PDGF released from platelets aggregating at sites of endothelial denudation may also penetrate into the lesion and reach the SMC. Prostaglandins, adenosine and other growth-inhibitory substances released from the endothelium, and adenosine from plasma and aggregating platelets counteract the effect of these mitogens. The final rate of SMC proliferation, and consequently lesion progression, is dependent on the balance between growth-promoting and growth-inhibiting substances. Proliferating SMC secrete collagen, elastin and proteoglycans to the surrounding matrix, thus giving rise to the characteristic fibrous plaque (Fig. 1D). The rate of intra- and extracellular lipid deposition in the lesions is enhanced by a high plasma concentration of LDL, a low plasma concentration of HDL and by a low number of LDL receptors per cell. If factors favouring SMC proliferation and intimal lipid deposition dominate the arterial environment, an irreversible atherosclerotic lesion is ultimately formed (Fig. 1E). At present, this hypothesis is based mainly on our experience of the regulation of metabolism and proliferation of arterial wall cells in culture. It is important to keep in mind that production of a PDGF-like mitogen this far has been demonstrated only in SMC isolated from rat arteries and that we still don’t know if human arterial SMC react in a similar way. It is possible that in the future improved immunological techniques and in situ hybridization may help us to identify the factors responsible for proliferation of SMC in human atherosclerotic lesions.

195 References 1 Holmes, D.R., Elveback, L.R., Frye, R.L., Kottke, B.A. and Ellefson, R.D., Association of risk factor variables and coronary artery disease documented with angiography, Circulation, 63 (1981) 293. 2 Millner, N.E., Hammet, F., Saltissi, S., Rao, S., van Zeller, H., Coltart, J. and Lewis, B., Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins, Brit. Med. J., 282 (1981) 1741. 3 Hamsten, A., Walldius, G., Szamosi, A., Dahlen, G. and de Faire. U., Relationship of angiographically defined coronary artery disease to serum lipoproteins and apoproteins in young survivors of myocardial infarction, Circulation, 73 (1986) 1097. 4 Ross, R. and Glomset, J.A., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369 and 420. 5 Reidy, M.A., A reassessment of endothelial injury and arterial lesion formation, Lab. Invest., 53 (1985) 513. 6 Seifert. R.A.. Schwartz, S.S. and Bowen-Pope, D.F., Developmentally regulated production of platelet-derived growth factor-like molecules, Nature (Lond.), 311 (1984) 669. 7 Nilsson, J.. Sjiilund, M., Palmberg, L., Thyberg, J. and Heldin. C.-H., Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein, Proc. Nat. Acad. Sci. (USA), 82 (1985) 4418. 8 Shimokado, K., Raines, E.W., Madtes, D.K., Barett, T.B., Benditt, E.P. and Ross, R., A significant part of macrophage-derived growth factor consists of at least two forms of PDGF, Cell, 43 (1985) 277. 9 DiCorletto, P.E. and Bowen-Pope, D.F., Cultured endothelial cells produce a platelet-derived growth factor-like protein, Proc. Nat. Acad. Sci. (USA), 80 (1983) 1919. 10 Gerrity, R.G.. The role of the monocyte in atherogenesis, Amer. J. Path., 103 (1981) 181 and 191. 11 Joris, I., Zand, T.. Nunnari, J.J., Krolikowski, F.J. and Majno, G., Studies on the pathogenesis of atherosclerosis, Amer. J. Path., 113 (1983) 341. 12 Faggiotto, A.. Ross, R. and Harker, L., Studies of hypercholesterolemia in the nonhuman primate, Part 1 (Changes that lead to fatty streak formation), Arteriosclerosis, 4 (1984) 323. 13 Faggiotto, A. and Ross, R., Studies of hypercholesterolemia in the nonhuman primate, Part 2 (Fatty streak conversion to fibrous plaque), Arteriosclerosis, 4 (1984) 341. 14 Florentin, R.A., Nam, SC., Lee, K.T. and Thomas, W.A., Increased 3H-thymidine incorporation into endothelial cells of swine fed cholesterol for 3 days, Exp. Mol. Path., 10 (1969) 250. 15 McGill, H.C., Persistent problems in the pathogenesis of atherosclerosis, Arteriosclerosis, 4 (1984) 443. 16 Thomas. W.A. and Kim, D.N., Atherosclerosis as hyperplastic and/or neoplastic disease, Lab. Invest., 48 (1983) 245. 17 Schwartz. S.M., Gajdusek. C.M. and Selden, SC., Vascu-

lar wall growth 18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

control

- The role of the endothelium,

Arteriosclerosis, 1 (1981) 107. Schwartz, SM. and Ross, R., Cellular proliferation in atherosclerosis and hypertension, Progr. Cardiovasc. Dis., 26 (1984) 355. Bondjers, G., Brattsand, R., Bylock, A., Hansson, G.K. and Bjbrkerud, S., Endothelial integrity and atherogenesis in rabbits with moderate hypercholesterolemia. Artery, 3 (1977) 395. Reidy, M.A. and Bowyer, D.E., Distortion of endothelial repair. The effect of hypercholesterolemia on regeneration of aortic endothelium following injury by endotoxin - A scanning electron microscope study, Atherosclerosis. 29 (1978) 459. Taylor, K., Glagov, S., Lamberti, J., Vesselinovitch, D. and Schaffner, T., Surface configuration of early atheromatous lesions in controlled-pressure perfusion-fixed monkey aortas, Scan. Electron Microsc., 2 (1978) 449. Bjarkerud, S., Reaction of the aortic wall of the rabbit after superficial, longitudinal, mechanical trauma, Virchows Arch. Path. Anat., 347 (1969) 197. Stemerman, M.B. and Ross, R., Experimental arteriosclerosis, Part 1 (Fibrous plaque formation in primates, an electron microscopic study), J. Exp. Med., 136 (1972) 769. Sheppard, B.L. and French, J.E., Platelet adhesion in the rabbit abdominal aorta following the removal of the endothelium - A scanning and transmission electron microscopical study, Proc. Roy. Sot. Lond. (B), 176 (1971) 427. Groves, H.M., Kinlough-Rathbone, R.L., Richardson, M., Moore, S. and Mustard, J.F., Platelet interaction with damaged rabbit aorta, Lab. Invest.. 40 (1979) 194. Bondjers, G. and Bjbrkerud, S., Arterial repair and atherosclerosis after mechanical injury, Part 2 (Tissue response after induction of a total local necrosis), Atherosclerosis, 14 (1971) 259. Poole, J.C.F., Cromwell, S.B. and Benditt, E.P., Behavior of smooth muscle cells and formation of extracellular structures in the reaction of arterial wall to injury, Amer. J. Path., 62 (1971) 391. Fishman, J.A., Ryan, G.B. and Karnovsky, M.J., Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening, Lab. Invest., 32 (1975) 339. Webster, W.S., Bishop, S.P. and Geer, J.C., Experimental aortic intimal thickening - Morphology and source of intimal cells, Amer. J. Path., 76 (1974) 245. Friedman, R.J., Moore, S. and Singal, D.P., Repeated endothelial injury and induction of atherosclerosis in normolipemic rabbits by human serum, Lab. Invest., 32 (1975) 404. Reidy, M.A. and Silver, M., Endothelial regeneration, Part 7 (Lack of intimal proliferation after defined injury to rat aorta), Amer. J. Path., 118 (1985) 173. Strong, J.P., Restrepo, C. and Gunman, M., Coronary and aortic atherosclerosis in New Orleans, Part 2 (Comparison of lesions by age, sex and race), Lab. Invest., 39 (1978) 364. Velican, D. and Velican, C., Atherosclerotic involvement

196

34

35

36

37

38

39

40

41 42

43

44

45

46

47

48

49

of the coronary arteries of adolescents and young adults, Atherosclerosis, 36 (1980) 449. Velican, C. and Velican, D., The precursors of coronary atherosclerotic plaques in subjects up to 40 years old, Atherosclerosis, 37 (1980) 33. Stary, H.C., Evolution of atherosclerotic plaques in the coronary arteries of young adults, Arteriosclerosis, 3 (1983) 471a. Ross, R., Wight, T.N., Strandness, E. and Thiele, B., Human atherosclerosis, Part 1 (Cell constitution and characteristics of advanced lesions of the superficial femoral artery), Amer. J. Path., 114 (1984) 79. Jonasson, L., Holm, J., Skalli, O., Bondjers, G. and Hansson, G.K., Regional accumulation of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque, Arteriosclerosis, 6 (1986) 131. Benditt, E.P. and Benditt, J.M., Evidence for a monoclonal origin of human atherosclerotic plaques,Proc. Nat. Acad. Sci. (USA), 70 (1973) 1753. Pearson, T.A., Wang, A., Solez, K. and Hepinstall, R.H., Clonal characteristics of fibrous plaque and fatty streaks from human aortas, Amer. J. Path., 81 (1975) 379. Thomas, W.A., Reiner, J.M., Janakidevi, K., Florentin, R.A. and Lee, K.T., Population dynamics of arterial cells during atherogenesis, Part 10 (Study of monotypism in atherosclerotic lesions of black women heterozygous for glucose-6-phosphate dehydrogenase), Exp. Mol. Path., 31 (1979) 367. Martin, G.S. and Sprague, C.A., Clonal senescence and atherosclerosis, Lancet, ii (1972) 1370. Schwartz, S.M., Reidy, M.R. and Clowes, A., Kinetics of atherosclerosis - A stem cell model, Ann. N.Y. Acad. Sci., 454 (1985) 301. Campbell, G.R. and Campbell, J.H., Smooth muscle phenotypic changes in arterial wall homeostasis - Implications for the pathogenesis of atherosclerosis, Exp. Mol. Path., 42 (1985) 139. Baumgartner, H.R. and Studer, A., Folgen des Gefasskatheterismus am normound hypercholesterolemischen Kaninchen, Path. Microbial., 29 (1966) 393. Stemerman, M.B. and Ross, R., Experimental atherosclerosis, Part 1 (Fibrous plaque formation in primates, an electron microscope study), J. Exp. Med., 136 (1972) 769. Owens, G.K. and Reidy, M.A., Hyperplastic growth response of vascular smooth muscle cells following induction of acute hypertension in rats by aortic coarctation, Circ. Res., 57 (1985) 695. Reidy, M.A. and Schwartz, S.M., Endothelial regeneration, Part 3 (Time course of intimal changes after small defined injury to rat aortic endothelium), Lab. Invest., 44 (1981) 301. Ramsey, M.M., Walker, L.N. and Bowyer, D.E., Narrow superficial injury to rabbit aortic endothelium - The healing process as observed by scanning electron microscopy, Atherosclerosis, 43 (1982) 233. Scott, R.F., Imai, H., Makita, T., Thomas, W.A. and Reiner, J.M., Lining cell and intimal smooth muscle cell

response and Evans blue staining in abdominal aorta of young swine after denudation with balloon catheter, Exp. Mol. Path., 33 (1980) 185. 50 Goldberg, I., Stemerman, M.B. and Handin, RI., Vascular permeation of platelet factor IV after endothelial injury, Science, 209 (1980) 610. 51 Clowes, A.W. and Schwartz, S.S., Significance of quiescent smooth muscle migration of the injured rat carotid artery, Circ. Res., 56 (1985) 139. 52 Walker, L.N., Ramsay, M.M. and Bowyer, D.E., Endothelial healing following defined injury to rabbit aorta Depth of injury and mode of repair, Atherosclerosis, 47 (1980) 123. 53 Schwartz, SM., Gajdusek, C.M., Reidy, M.A., Selden, S.C. and Haudenschild, C.C., Maintenance of integrity in aortic endothelium, Fed. Proc., 39 (1980) 2618. 54 Schwartz, S.M. and Benditt, E.P., Aortic endothelia cell replication, Part 1 (Effects of age and hypertension in rat), Circ. Res., 41 (1977) 248. 55 Kunz, J., Schreiter, B., Schubert, B., Voss, K. and Krieg, K., Experimentelle Untersuchungen tiber die Regeneration der Aortenendothelzellen, Acta Histochem., 61s (1978) 53. 56 Gerrity, R.G., Naito, H.K., Richardson, M. and Schwartz, C.J., Dietary induced atherogenesis in swine, Amer. J. Path., 95 (1979) 775. 57 Thomas, W.A., Lee, K.T. and Kim, D.N., Cell population kinetics in atherogenesis - Cell births and losses in intimal cell mass-derived lesions in the abdominal aorta of swine, Ann. Acad. N.Y. Sci., 454 (1985) 305. 58 Nilsson, J., Svensson, J., Hamsten, A. and de Faire, U., Increased platelet-derived mitogenic activity in plasma of young patients with coronary atherosclerosis, Atherosclerosis, 61 (1986) 237. 59 Gerrity, R.G., Goss, J.A. and Soby, L., Control of monocyte recruitment by chemotactic factor(s) in lesion-prone areas of swine aorta, Arteriosclerosis, 5 (1985) 55. 60 Farber, H.W., Center, D.M. and Rounds, S., Bovine and human endothelial cell production of neutrophil chemoattractant activity in response to components of the angiotensin system, Circ. Res., 57 (1985) 898. 61 Bevilacqua, M.P., Pober, J.S., Wheeler, M.E., Cotran, R.S. and Gimbrone, M.A., Interleukin 1 acts on cultured vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes and related leukocyte cell lines, J. Clin. Invest., 76 (1985) 2003. 62 Hansson, G.K., Bondjers, G., Bylock, A. and Hjalmarsson, L., Ultrastructural studies on the localization of IgG in the aortic endothelium and subendothelial intima of atherosclerotic and nonatherosclerotic rabbits, Exp. Mol. Path., 33 (1980) 302. 63 Quinn, M.T., Parthasarathy, S. and Steinberg, D., Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoproteins, Proc. Nat. Acad. Sci. (USA), 82 (1985) 5949. 64 Poole, J.C.F., Cromwell, S.B. and Benditt, E.P., Behaviour of smooth muscle cells and formation of extracellular

197

65

66 67

68

69

70

71

72

73

74

75 76

77

78

79

structures in the reaction of arterial wall to injury, Amer. J. Path., 62 (1971) 391. Mosse. P.R.L., Campbell, G.R., Wang, Z.L. and Campbell, J.H., Smooth muscle phenotype expression in human carotid arteries, Part 1 (Comparison of cells from diffuse intimal thickenings adjacent to atheromatous plaques with those of the media), Lab. Invest., 53 (1985) 556. Charnley-Campbell, .I., Campbell, G.R. and Ross, R., The smooth muscle cell in culture, Physiol. Rev., 59 (1979) 1. Charnley-Campbell, J.H., Campbell, G.R. and Ross, R., Phenotype-dependent response of cultured aortic smooth muscle to serum mitogeng J. Cell Biol., 89 (1981) 379. Thyberg, J., Palmberg, L., Nilsson, J., Ksiazek, T. and Sjolund, M., Phenotype modulation in primary cultures of arterial smooth muscle cells - On the role of platelet-derived growth factor, Differentiation, 25 (1983) 156. Campbell, G.R. and Campbell, J.H., Smooth muscle phenotypic changes in arterial wall homeostasis - Implications for the pathogenesis of atherosclerosis, Exp. Mol. Path., 42 (1985) 139. Thyberg, J., Nilsson, J., Palmberg, L. and Sjiilund, M., Adult human arterial smooth muscle cells in primary culture - Modulation from contractile to synthetic phenotype, Cell Tissue Res., 239 (1985) 69. Charnley-Campbell, J.H. and Campbell, G.R., What controls smooth muscle phenotype? Atherosclerosis, 40 (1981) 347. SJolund, M., Nilsson, J., Palmberg, L. and Thyberg, J., Phenotype modulation in primary cultures of arterial smooth-muscle cells - Dual effect of prostaglandin Et, Differentiation, 27 (1984) 158. Castellot, J.J., Favreau, L.V., Karnovsky, M.J. and Rosenberg, R. D., Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin - Possible role of a platelet endoglycosidase, J. Biol. Chem., 257 (1982) 11256. Heldin, C-H., Wasteson, A. and Westermark, B., Platelet-derived growth factor, Mol. Cell. Endocrinol., 39 (1985) 169. Bishop, M.J., Cellular oncogenes and retrovituses, Ann. Rev. Biochem., 52 (1983) 301. Waterfield, M.D., Scrace, G.T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H.. Huang, J.S. and Deuel, T.F., Platelet-derived growth factor is structurally related to the putative transforming protein p28’~“” of simian sarcoma virus, Nature (Lond.), 304 (1983) 35. Doolittle, R.F., Hunkapiller, M.W., Hood, L.E., Devare, S.G., Robbins, K.C., Aaronson, SE. and Antoniades, H.A., Simian sarcoma virus oncogene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor, Science, 221 (1983) 275. Johnsson. A., Heldin, C.-H., Wasteson, A., Westermark, B., Deuel, T.F., Huang, J.S., Seeburg, D.H., Gray, E., Ulhich, A., Scrace, G., Stroobant, P. and Waterfield, M.D., The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor, EMBO J., 3 (1984) 921. Deuel, T.F., Huang, J.S.. Huang, S.S., Stroobant, P. and

80

81

82

83

84

85

86

87

88

89

90

91

Waterfield, M.D., Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells, Science, 221 (1983) 1348. Owen, A.J., Pantazis, P. and Antoniades, H.N., Simian sarcoma virus-transformed cells secrete a mitogen identical to platelet-derived growth factor. Science, 225 (1984) 54. Betsholtz, C., Heldin, C.-H., Nister, M., Ek, B., Wasteson, A. and Westermark, B., Synthesis of a PDGF-like growth factor in human glioma and sarcoma cells suggest the expression of the cellular homologue to the transforming protein of simian sarcoma virus, Biochem. Biophys. Res. Commun., 117 (1983) 176. Pantazis, P., Pelicci, P.G., Dalla-Favera, R. and Antoniades, H.N., Synthesis and secretion of proteins resembling platelets derived growth factor by human glioblastoma and fibrosarcoma cells in culture, Proc. Nat. Acad. Sci. (USA), 82 (1985) 2404. Syms, A.J., Norris, J.S. and Smith, R.G., Autocrine regulation of growth, Part 1 (Glucocorticoid inhibition is overcome by exogenous platelet-derived growth factor), Biochem. Biophys. Res. Commun., 122 (1984) 68. Norris, J.S., Cornett, L.E., Hardin, J.W., Kohler, P.H., Mac-Leod, S.L., Srivastava, A., Syms, A.L. and Smith, R.G., Autocrine regulation of growth, Part 2 (Glucocorticoids inhibit transcription of c-sis oncogene-specific RNA transcripts), Biochem. Biophys. Res. Commun.. 122 (1984) 124. Sejersen, T., Betzholtz, C., Sjijlund, M.. Heldin, C.-H., Westermark, B. and Thyberg, J., Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A-chain but not for the B-chain (c-sis) of PDGF and produce a PDGF-like protein, Proc. Nat. Acad. Sd. (USA), In press. Walker, L.N., Bowen-Pope, D.F. and Reidy, M.A., Secretion of platelet-derived growth factor-like activity by arterial smooth muscle cells is induced as a response to injury, J. Cell Biol., 99 (1984) 416a. Grtinwald, J. and Haudeschild, C.C., Intimal injury in vivo activates vascular smooth muscle cell migration and explant outgrowth in vitro, Arteriosclerosis, 4 (1984) 153. Pietila, K. and Nikkari, T., Enhanced growth of smooth muscle cells from atherosclerotic rabbit aortas in culture, Atherosclerosis, 36 (1980) 241. Stavenow, L., Differences in bovine aortic smooth muscle cells cultured from spontaneous atherosclerotic lesions of different severity within the same vessel. Atherosclerosis, 53 (1984) 337. Orekov, A.N., Kosykh, V.A., Repin, V.S. and Smirnov, V.N., Cell proliferation in normal and atherosclerotic human aorta, Part 2 (Autoradiographic observation on deoxyribonucleic acid synthesis in primary cell culture), Lab. Invest., 48 (1983) 749. Nakao, J., Ooyama, T., Chang, W.C., Murota, S. and Orimo, H., Platelets stimulate aortic smooth muscle cell migration in vitro - Involvement of 12-L-hydroxy5,8,10,14-eicosatetranoic acid, Atherosclerosis, 43 (1982) 143.

198 92 Leibovich, S. and Ross, R., A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro, Amer. J. Path., 84 (1976) 501. 93 Glenn, K.C. and Ross, R., Human monocyte-derived growth factor(s) for mesenchymal cells - Activation of secretion by endotoxin and concanavalin A, Cell, 25 (1981) 603. 94 Diegehnann, R.F., Cohen, I.K. and Kaplan, A.M., Effects of macrophages on fibroblast DNA synthesis and proliferation, Proc. Sot. Exp. Biol. Med., 169 (1982). 95 Leibovich, S.J. and Ross, R., The role of the macrophage in wound repair - A study with hydrocortisone and antimacrophage serum, Amer. J. Path., 78 (1975) 71. 96 Barrett, T.B., Gajdusek, C.M., Schwartz, S.M., McDougall, J.K. and Benditt, E.P., Expression of the sis gene by endothelial cells in culture and in vivo, Proc. Nat. Acad. Sci. (USA), 81 (1984) 6772. 97 Jaye, M., McConathy, E., Drohan, W., Tong, B., Deuel, T. and Maciag, T., Modulation of the sis gene transcript during endothelial differentiation in vitro, Science, 22 (1985) 882. 98 Kazlauskas, A. and DiCorleto, P.E., Cultured endothelial cells do not respond to platelet-derived growth factor like protein in an autocrine manner, Biochim. Biophys. Acta, 846 (1985) 405. 99 DiCorleto, P., Cultured endothelial cells produce multiple growth factors for corrective tissue cells, Exp. Cell Res., 153 (1984) 167. 100 Van Buul-Wortelboer, M.F., Brinkman, H.J.M., Dingemans, K.P., De Groot, P.G., Van Aken, W.G. and Van Mourik, J.A., Reconstitution of the vascular wall in vitro - A novel model to study interactions between endothelial and smooth muscle cells, Exp. Cell Res., 162 (1986) 151. 101 Pearson, J.D., Hellewell, P.G. and Gordon, J.L., Adenosine uptake and adenosine nucleotide metabolism by vascular endothelium. In: R.M. Beme, T.W. Rall and R. Rubio (Eds.), Regulatory Functions of Adenosine (Developments of Pharmacology, Vol. 2), Martinus Nijhoff, Hague, 1983, pp. 333-345. 102 Jonzon, B., Nilsson, J. and Fredholm, B.B., Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells, J. Cell Physiol., 124 (1985) 451. 103 Nilsson, J. and Olsson, A.G., Prostaglandin Ei inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor, Atherosclerosis, 53 (1984) 77. 104 Castellot, J.J., Addonizio, M.L., Rosenberg, R.D. and Kamovsky, M.J., Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth, J. Cell Biol., 90 (1981) 372. 105 Hoover, R.L., Rosenberg, L.D., Haering, W. and Kamovsky, M.J., Inhibition of rat arterial smooth muscle cell proliferation by heparin, Part 2 (In vitro studies), Circ. Res., 47 (1980) 578. 106 Clowes, A.W. and Karnovsky, M.J., Suppression by heparin of smooth muscle cell proliferation of injured arteries, Nature (Lond.), 265 (1977) 625.

107 Castellot, 3.5. et al., Binding and internalization of heparin by vascular smooth muscle cells, J. Cell. Physiol., 124 (1985) 13. 108 Castellot, J.J., Cochran, D.L. and Kamovsky, M.J., Effect of heparin on vascular smooth muscle cells, Part 1 (Cell metabolism), J. Cell Physiol., 124 (1985) 21. 109 Kannel, W.B., Schwartz, M.J. and McNamar, P.M., Blood pressure and the risk of coronary heart disease - The Framingham study, Dis. Chest., 56 (1969) 43. 110 Kannel, W.B. et al., Optimal resources for primary prevention of atherosclerotic diseases, Circulation, 70 (1984) 157. 111 Hadjiisky, P. and Peyri, N., Hypertensive arterial disease and atherogenesis, Part 1 (Intimal changes in the old spontaneously hypertensive rat), Atherosclerosis, 44 (1982) 181. 112 Bevan, R.D., van Marthens, E. and Bevan, J., Hyperplasia of vascular smooth muscle in experimental hypertension in the rabbit, Circ. Res., 38 (1976) 58. 113 Owens, G.K., Differential effects of antihypertensive drug therapy on vascular smooth muscle cell hypertrophy, hyperploidy, and hyperplasia in the spontaneously hypertensive rat, Circ. Res., 56 (1985) 525. 114 Owens, G.K., Rabinovitch, P.S. and Schwartz, S.M., Smooth muscle cell hypertrophy versus hyperplasia in hypertension, Proc. Nat. Acad. Sci. (USA), 78 (1981) 7759. 115 Campbell-Boswell, M. and Robertson, A.L., Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro, Exp. Mol. Path., 35 (1981) 265. 116 Blaes, N. and Boissel, J.-P., Growth-stimulating effect of catecholamines on rat aortic smooth muscle cells in culture, J. Cell. Physiol., 116 (1983) 167. 117 Berk, B.C., Alexander, R.W., Brock, T.A., Gimbrone, M.A. and Webb, R.C., Vasoconstriction - A new activity for platelet-derived growth factor, Science, 232 (1986) 87. 118 Alexander, R.W., Brock, T.A., Gimbrone, M.A. and Rittenhouse, S.E., Angiotensin increases inositol triphosphate and calcium in vascular smooth muscle, Hypertension, 7 (1985) 447. 119 Charest, R., Prpic, V., Exton, J.H. and Blackmore, P.F., Stimulation in inositol triphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca*+, Biochem. J., 227 (1985) 79. 120 Berridge, M.J. and Irvine, R.F., Inositol triphosphate, a novel second messenger in cellular signal transduction, Nature (Lond.), 312 (1984) 315. 121 Betz, E., HZmmerle, D. and Viele, A., Ca2+-entry blockers and atherosclerosis, Int. J. Angiol., 3 (1984) 33. 122 Nilsson, J., Sjiilund, M., Palmberg, L., von Euler, A.M., Jonzon, B. and Thyberg, J., The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation, Atherosclerosis, 58 (1985) 109. 123 Nilsson, J., von Euler, A.M. and Dalsgaard, C.-J., Stimulation of connective tissue cell growth by substance P and substance K, Nature (Lond.), 315 (1985) 61. 124 Cocks, T.M., Angus, J.A., Campbell, J.H. and Campbell,

199

125

126

127

128

129

G.R., Release and properties of endothelium-derived relaxing factor, .I. Cell. Physiol., 123 (1985) 310. Haudenschild, C.C., Grtinwald, .I. and Chobanian, A.V.. Effects of hypertension on migration and proliferation of smooth muscle cells in culture, Hypertension, 7 (1985) I-101. Gordon, T., Kannel, W.B., Castelli, W.B. and Dawber, T.R., Lipoproteins, cardiovascular disease and death The Framingham study, Arch. Int. Med., 141 (1981) 1128. Carlson, L.A., Serum lipids and atherosclerotic disease. In: L.A. Carlson and B. Pernow (Eds.), Metabolic Risk Factors in Ischemic Heart Disease, Raven Press, New York, 1982, pp. 1-15. Goldstein, J.L. and Brown, M.S., The low-density lipoprotein pathway and its relation to atherosclerosis, Ann. Rev. Biochem., 46 (1977) 897. Brown, MS., Kovanen, P.T. and Goldstein, J.S., Regulation of plasma cholesterol by lipoprotein receptors, Science, 212 (1981) 628.

130 Brown, M.S. and Goldstein, J.L., Lipoprotein metabolism in the macrophage - Implications for cholesterol deposition in atherosclerosis, Ann. Rev. B&hem., 52 (1983) 223. 131 Hemiksen, T., Mahoney, E.M. and Steinberg, D., Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells Recognition by the receptor for acetylated lipoproteins, Proc. Nat. Acad. Sci. (USA), 78 (1981) 6499. 132 Parthasarathy, S., Steinbrecher, U.P., Bamett, J., Witztum. J.L. and Steinberg, D., Essential role of phospholipase A, activity in endothelial-induced modification of low-density lipoprotein, Proc. Nat. Acad. Sci. (USA), 82 (1985) 3000. 133 Libby, P., Miao, P., Ordovas, J.M. and Schaefer, E.J., Lipoproteins increase growth of mitogen-stimulated arterial smooth muscle cells, J. Cell. Physiol.. 124 (1985) 1. 135 Harker, L.A. and Hazzard, W., Platelet kinetic studies in patients with hyperlipoproteinemia: effect of clofibrate therapy, Circulation, 60 (1979) 492.