During recent years, it has become increasingly evident

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1 Review Regulation of Differentiated Properties and Proliferation of Arterial Smooth Muscle Cells Johan Thyberg, Ulf Hedin, Maria Sjolund, Lena Palmberg, and Bradford A. Bottger During recent years, it has become increasingly evident that arterial smooth muscle cells occur in at least two distinct states, usually referred to as a synthetic and a contractile phenotype. Synthetic-state cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. They are found in the embryo and the young growing organism, where they take part in the formation of the vessel wall. Contractile-state cells have a musclelike appearance and contract in response to chemical and mechanical stimuli. They predominate in the vessels of adults and are primarily involved in the control of blood pressure and flow. However, these cells are able to return to a synthetic phenotype, and this appears to be an important early event in atherogenesis. This review briefly summarizes the present knowledge concerning the regulation of differentiated properties and proliferation of arterial smooth muscle cells. Most of the discussion will deal with studies on cultured cells, which so far are the most abundant. Particular attention will be paid to the role of extracellular matrix components like fibronectin and laminin and of polypeptide mitogens like platelet-derived growth factor (PDGF). It is believed that future studies in this area will help to widen our understanding of vasculogenesis and the initial stages in the pathogenesis of atherosclerosis. Development of Arterial Smooth Muscle Cells In the embryo, the large arteries arise from the mesoderm. 123 Mesenchymal cells initially form a lining of endothelial cells around spaces filled with fluid. Subsequently, additional mesenchymal cells accumulate and start to differentiate into smooth muscle cells. The signals that control this process are largely unknown. During most of the fetal and early postnatal period, the smooth muscle cells have a fibroblast-like appearance with an extensive rough endoplasmic reticulum, a prominent Golgi complex, and only a few myofilaments. 45 They From the Department of Medical Cell Biology, Karolinska Institute, Stockholm, Sweden. This work was supported by grants from the Swedish Medical Research Council, the Swedish Heart Lung Foundation, and the King Gustaf V 80th Birthday Fund. Address for correspondence: Dr. Johan Thyberg, Department of Medical Cell Biology, Karolinska Institute, Box 60400, S Stockholm, Sweden. Received May 22, 1990; revision accepted July 31, proliferate and secrete extracellular matrix components like collagen and elastin. 678 At this stage, /3-actin is the main actin isoform and vimentin is the main intermediate filament protein Later on, as the vessels mature, the synthetic organelles decrease in size and myofilaments occupy successively larger parts of the cytoplasm. 45 This is associated with a decrease in the synthetic and proliferative activities, and instead the ability to contract is established. In parallel, there is a decreased /3-actin expression and an increased expression of smooth muscle-specific a-actin. 9 ' Moreover, the content of the intermediate filament protein desmin and the fraction of cells that show a positive staining for both vimentin and desmin increase There is also a distinct rise in the cellular content of myosin and tropomyosin In adults, the arterial media is made up of highly differentiated smooth muscle cells arranged in concentric layers Each cell is encircled by a basement membrane composed of collagen type IV, laminin, entactin (nidogen), and heparan sulfate proteoglycans This macromolecular network plays an important role in the homeostasis of the cells by direct interaction with them, by regulating the transport of molecules to and from them, and by establishing a link with the surrounding extracellular matrix. The latter is primarily made up of elastic laminae, 2526 fibrils of collagen type I and III, 2728 and chondroitin sulfate and dermatan sulfate proteoglycans In short, the above changes in structure and function of arterial smooth muscle cells during development can be referred to as a shift from a synthetic to a contractile phenotype (Figure 1). To a large extent, this process resembles the conversion of proliferating myoblasts into postmitotic myotubes during the formation of striated muscle. 31 However, in contrast to these cells, the smooth muscle cells are able to return from a contractile to a synthetic phenotype. Most prominently, this takes place in connection with atherogenesis and when smooth muscle cells are established in culture Since the cells appear to be able to shift back and forth between these states and do not return to a multipotential state, it has been suggested that the process should be referred to as phenotypic modulation rather than dedifferentiation. 33 In the following, we use the term phenotypic modulation to describe some major variations in cellular behavior within the state of arterial smooth muscle differentiation. 966

2 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 967 Occurrence SYNTHETIC PHENOTYPE Structure Function embryo and young growing organism atherosclerotic lesions euchromatic nucleus prominent ER and Golgi complex proliferation synthesis and secretion of extracellular matrix components normal development ITatherogenesis adult organism CONTRACTILE PHENOTYPE heterochromatic nucleus abundant actin and myosin filaments contraction in response to chemical and mechanical stimuli Figure 1. Schematic overview of changes in the differentiated properties of arterial smooth muscle cells during normal development and atherogenesis. Modulation of Smooth Muscle Phenotype during Atherogenesis For a general survey of the atherosclerotic process, the reader is referred to recent reviews In the early stages of the disease, smooth muscle cells migrate from the media to the intima of the arterial wall, where they proliferate and deposit extracellular matrix components, thereby forming a lesion that protrudes into the vessel lumen. Immunocytochemical studies with cell type-specific monoclonal antibodies have indicated that the lesions also contain numerous macrophages and T lymphocytes It is believed that a damage to the endothelial barrier may initiate the process. The smooth muscle cells are thus under the influence of a large variety of factors, not only blood components but also substances released by endothelial cells, platelets adhering to exposed subendothelial tissue, as well as macrophages and T lymphocytes invading the vessel wall. In addition, the smooth muscle cells may produce and release substances that affect their own function. Electron microscopic studies have revealed that the smooth muscle cells in atherosclerotic lesions are modified structurally, with a decrease in the content of myofilaments and an increase in the size of the rough endoplasmic reticulum and the Golgi complex Likewise, structural and functional changes indicating a modulation of the smooth muscle cells from a contractile to a synthetic phenotype have been observed in experimental animals after induction of intimal lesions by balloon catheterization ' 48 The shift in differentiated properties of the smooth muscle cells during atherogenesis and experimental lesion formation is also reflected in the expression of cytoskeletal proteins. In both cases, there is a switch from smooth muscle-specific a-actin to /3-actin and a decrease in the expression of smooth muscle myosin. 54 Moreover, the expression of desmin decreases, 5556 whereas the cells start to express low levels of cytokeratins. 57 Another sign of a changed smooth muscle phenotype during atherogenesis and experimental lesion formation is the expression of class II transplantation antigens ' 60 Further, monoclonal antibodies have been produced that react with surface antigens present on smooth muscle cells in intimal lesions but not in the normal media In Vitro Cultivation as Experimental System to Study Regulation of Smooth Muscle Phenotype Because of the great complexity of the in vivo environment, cell culture has become an important model for studies on the control of smooth muscle phenotype and proliferation. The main advantage of cell culture is the possibility to set up defined conditions and to study the effects of individual factors without the complex interplay with other cell types and without the hormonal and neuronal influences that exist in the intact organism. The first attempts to culture smooth muscle were made in the beginning of this century However, it was not until the early 1970s that this technique became well established. From then on, it has found use in a large number of investigations and has been described in recent reviews In this section, we will mainly deal with the shift in phenotype of adult arterial smooth muscle cells in primary culture. Cellular Fine Structure Fritz et al. 67 were the first to point out that during the first few days in primary culture arterial smooth muscle cells change from a muscle-like to a fibroblast-like morphology and that this precedes the onset of cellular proliferation. Similar findings were subsequently made in studies on smooth muscle cells from experimental animals as well as humans In short, the following sequence of events is observed (see Figure 1). Cells isolated by enzymatic digestion of the arterial media have a heterochromatic nucleus and a cytoplasm dominated by bundles of myofilaments coalescing in dense bodies and abundant mitochondria (Figure 2A). Initially, these cells adhere to the bottom of the culture dishes and then spread out over a period of a few days. At the same time,

3 968 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER 1990 M a r B. A\- M?o

4 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 969 Figure 2. Changes in fine structure during phenotypic modulation in primary culture. Smooth muscle cells were isolated enzymatically from the media of rat aorta, were grown in plastic dishes in nutrient medium containing 10% serum, and were fixed for transmission electron microscopy after varying times. A. Freshly isolated cell with a heterochromatic nucleus (N) and a cytoplasm dominated by myofilament bundles (F) and mitochondria (M)-a contractile phenotype. B. Two-day-old culture. Numerous free polyribosomes (R) and cisternae of rough endoplasmic reticulum (RER) appear in the perinuclear cytoplasm. Myofilament bundles (F) remain in the more peripheral cytoplasm an intermediate stage. C. Four-day-old culture. Euchromatic nucleus (N) and a cytoplasm dominated bycisternae of rough endoplasmic reticulum (RER) and a large juxtanuclear Golgi complex (G) -a synthetic phenotype. Bars=0.5 fim. there is an extensive reorganization of the cytoskeleton with loss of myofilament bundles and formation of actincontaining stress fibers and radiating networks of microtubules and intermediate filaments. 72 Moreover, cisternae of rough endoplasmic reticulum grow out from the nuclear envelope and a large juxtanuclear Golgi complex is formed. 70 In this way, a successive rebuilding of the cytoplasm occurs (Figures 2B and 2C). This process was found to take place independently of whether the culture medium is supplemented with whole blood serum or with plasma-derived serum. Cytoskeletal Markers As part of the phenotypic modulation of arterial smooth muscle cells, there is a characteristic shift in the expression of actin. The a-isoform predominates in freshly isolated, contractile cells, but increased amounts of the /3-isoform, as well as noticeable amounts of the y-isoform, are found in synthetic cells At least partly, this appears to be due to changes in actin mrna levels However, both in primary cultures and in passaged cells, the control of actin expression is still incompletely understood. It depends on growth rate as well as cell density and evidently includes both transcriptional/posttranscriptional and translational/ posttranslational events Although some variation in the published results exists, the main conclusion is that smooth muscle a-actin mrna and protein synthesis are suppressed during the phenotypic modulation and in actively proliferating cultures and increase again as the cultures become confluent and cell replication ceases. For the main part, nonmuscle /3-actin behaves in the opposite way. In any case, smooth muscle a-actin is a marker for vascular smooth muscle cells not only in primary cultures but also in subcultures and in established cell lines On the other hand, it is not a good tool to distinguish smooth muscle cells in a contractile phenotype from cells in a synthetic phenotype. In other cytoskeletal components, the phenotypic modulation in primary culture is associated with a distinctly decreased expression of smooth muscle myosin and an increased expression of nonmuscle myosin; as in the case of actin, the expression of myosin is also influenced by the density and growth state of the cultures The cellular content of tropomyosin decreases 7498 and the content of desmin decreases, whereas the content of vimentin increases These changes in cytoskeletal composition are all indicative of a change from a highly differentiated smooth muscle phenotype to a more fibroblast-like phenotype. Activation of Synthetic and Secretory Functions In parallel with the structural transformation of the smooth muscle cells in primary culture, overall RNA and protein synthesis and cellular proliferation are activated Moreover, synthesis and secretion of extracellular matrix components like collagen, elastin, and proteoglycans are enhanced Type I is the main collagen species produced, together with smaller amounts of type III and V. Immunocytochemical studies have indicated that the smooth muscle cells further secrete laminin and type IV collagen and surround themselves with a basement membrane-like layer. 100 Neonatal smooth muscle cells, which are already in a synthetic phenotype in vivo, have likewise been described as secreting collagen, elastin, and proteoglycans in primary culture As outlined in previous reviews, all these macromolecules are also produced by subcultured smooth muscle cells Lipid Metabolism Studies on rabbit aortic smooth muscle cells in primary culture have demonstrated that the modulation into a synthetic phenotype is associated with increased binding and degradation of very low density lipoprotein (VLDL) and increased binding of low density lipoprotein (LDL); in both cases, the binding appeared to be mediated via the apolipoprotein (apo) B/E receptor Conceivably, this reflects an increased need of cholesterol and other lipid components for membrane synthesis in the growing and rapidly proliferating synthetic-state cells. In an investigation on subcultured rat aortic smooth muscle cells, it was found that cells containing only vimentin accumulated more LDL than cells containing both vimentin and desmin. On the other hand, there was no difference in ingestion of LDL between cells containing and those lacking smooth muscle a-actin. Cells cultured in the presence of 10% serum took up more LDL than cells cultured in the presence of 1% serum. 110 These results support the idea that the ability of smooth muscle cells to ingest and metabolize lipoproteins is correlated with their phenotypic state and replicative activity. Role of Extracellular Matrix Components As mentioned above, it was noted that the phenotypic modulation of arterial smooth muscle cells in primary culture takes place equally well in media containing whole blood serum or plasma-derived serum This suggested that one or several plasma components may be involved. When exploring this possibility, it was found that a substrate of the plasma protein fibronectin efficiently promotes the transition of rat aortic smooth muscle cells from a contractile to a synthetic phenotype during primary culture in a serum-free medium Under these conditions, the cells passed through the morphological transformation described previously, and RNA and protein synthesis were activated. In contrast, DNA replication and cell proliferation did not start until the cultures had been exposed to serum or PDGF. These

5 970 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER 1990 CONTRACTILE PHENOTYPE SYNTHETIC PHENOTYPE nucleus actin filament bundles ER nucleus Golgi integrins no initial change in phenotype rapid change in phenotype fibronectin substrate slow change in phenotype due to endogenous secretion of fibronectin actin filament bundles iiiiiiiiiiiiiiniiiiniiiniiiimuitiiiiniiiiniiiniiiiiiiiiitiiii laminin substrate Figure 3. Schematic representation of the role of fibronectin and laminin in the phenotypic modulation of arterial smooth muscle cells. findings confirm the notion that the phenotypic modulation is a necessary, but not sufficient, requirement for smooth muscle proliferation in vitro Moreover, they demonstrate that it is possible to study the change in overall differentiated properties and the onset of cell proliferation separately. The results agree with the established role of fibronectin in cell attachment and spreading and in supporting the ability of cells to respond to growth factors and proliferate. 113 " 116 The power of fibronectin to bring about the change in phenotype of the smooth muscle cells was shown to reside in the cell-binding domain of the molecule, whereas the collagen- and heparin-binding domains were inactive in this respect. 112 Further, the transition of the cells into a synthetic phenotype was distinctly slower on substrates of laminin and type IV collagen, two major components of the basement membrane that normally surrounds smooth muscle, than on a substrate of fibronectin. 112 However, with time an increasing proportion of the cells turned into a synthetic phenotype, apparently due to endogenous secretion of fibronectin. In support of this conclusion, it was found that cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. 112 This suggests that the cells are able to sense the macromolecular composition of the pericellular matrix and to adjust their biosynthetic activities thereafter. Similar conclusions have been drawn in studies on subcultured cells. 117 Next, it was demonstrated that the interaction between the smooth muscle cells and fibronectin is mediated via a cell surface receptor belonging to the integrin superfamily of proteins (for reviews, see references , ) and the minimal cell-attachment sequence of fibronectin, Arg-Gly-Asp (RGD) Cells seeded on a substrate of an RGD-containing peptide (GRGDSC) attached and spread equally well as cells seeded on a substrate of the cell-binding domain of fibronectin or of intact fibronectin. Likewise, the overall change in cell morphology proceeded at similar rates on the three substrates. These processes were all distinctly slowed down by antibodies against the /3, integrin subunit (this antibody interfered with the attachment of the cells to fibronectin but not to vitronectin, another adhesive plasma protein that contains an RGD sequence). 122 The fibronectin receptors of the smooth muscle cells were isolated by sequential chromatography on columns of wheat germ agglutinin (WGA) and the cell-binding domain of fibronectin. 123 They were composed of two proteins with electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels of 160 and 115 kda under nonreducing conditions and of 150 and 130 kda under reducing conditions. Subsequent immunochemical and immunocytochemical studies have indicated that these molecules correspond to the 05- and /3,-subunits of the fibronectin receptor (unpublished observations). 124 The receptor molecules are arranged in linear arrays on the cell surface. They are co-aligned with, and presumably establish a transmembrane linkage between, fibronectin fibrils in the pericellular matrix and actin filament bundles in the cytoplasm During primary culture on a substrate of fibronectin, the receptors seem to primarily interact with bundles of nonmuscle actin. At the same time, the cells do not express mrna for smooth muscle a-actin, but the level of this actin isoform decreases only slowly, showing a partly diffuse staining in the cytoplasm. In subcultures, smooth muscle a-actin is again incorporated in actin filament bundles co-aligned with fibronectin receptors in the plasma membrane. 124 Summing up, these findings suggest important and diverse roles of fibronectin and laminin in controlling the

6 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 971 differentiated properties of arterial smooth muscle cells. Fibronectin promotes conversion of the cells from a contractile to a synthetic phenotype, whereas laminin acts in the opposite direction (Figure 3). There also exists circumstantial evidence indicating a role for fibronectin in the control of smooth muscle phenotype in vivo. In the chick embryo, fibronectin was found to appear before laminin in forming blood vessels, and the expression of laminin was suggested to be an early indicator of vascular maturation.125 Further, increased amounts of fibronectin appear in early atherosclerotic lesions Similar observations have been made in studies on the development of skeletal muscle. Fibronectin promotes proliferation of myoblasts and inhibits terminal differentiation and myotube formation, whereas laminin has the reverse effect '134 In addition, the synthesis and distribution of fibronectin and laminin changes during myogenesis The mechanisms of action behind the effects described above are not known. In part, adhesive molecules exert their effect by organization of the actin cytoskeleton. It is evident how this could explain the attachment and spreading of the cells on the substrate It has also been suggested that extracellular matrix components may influence gene expression via the effect on cell shape However, it is not clear how the contact with a fibronectin substrate brings about the extensive overall structural and functional reorganization of the smooth muscle cells. Moreover, there are evidently distinct differences in the effects of different extracellular matrix components. Although a substrate of laminin supported attachment and spreading of the smooth muscle cells, its effect on cellular phenotype was opposite to that of fibronectin.112 Therefore, cell shape alone is unlikely to be responsible. The elucidation of the signal transduction system that mediates the effect of extracellular matrix components on the differentiated properties of cells remains a challenge for the future. Although few experimental data are available to date, several other adhesive proteins may be important in the control of smooth muscle phenotype These include vitronectin, a 75 kda glycoprotein found in blood plasma and tissues This molecule contains an RGD sequence but binds to a cell surface receptor distinct from the fibronectin receptor,147 and supports attachment and spreading of freshly isolated smooth muscle cells less efficiently than fibronectin.122 Thrombospondin is a 450 kda trimeric glycoprotein stored in platelet a-granules and released during platelet degranulation. It is synthesized by a number of normal and transformed cells in culture and shows binding affinities for a variety of plasma and extracellular matrix proteins As discussed further below, this molecule has so far mainly been studied in relation to smooth muscle cell proliferation. Preliminary experiments suggest that it is not a good attachment substrate for these cells (unpublished observations). Tenascin is a large hexameric glycoprotein of the extracellular matrix, especially prominent during embryonic development and in tumors Among others, it has been found to interfere with the action of fibronectin Initial immunocytochemical studies show that tenascin is distinctly Figure 4. Subcultured rat aortic smooth muscle cells were detached by trypsinization and were reseeded in serum-free medium on substrates of fibronectin (A) or laminin plus type IV collagen (B). After 2 days of culture, the cells were fixed in buffered formaldehyde and were stained for indirect immunofluorescence microscopy with primary antibodies against smooth muscle a-actin. Bars=50 Aim.

7 972 ARTERIOSCLEROSIS VOL. 10, No 6, NOVEMBER/DECEMBER 1990 M N L* M N e> RER M IM * M " ' # / RER 1 B C Figure 5. Subcultured rat aortic smooth muscle cells were detached by trypsinization and were reseeded in serum-free medium on substrates of fibronectin (A) or laminin plus type IV collagen (B, C). After two days of culture, the cells were fixed in buffered glutaraldehyde and were processed for transmission electron microscopy. The cells cultured on fibronectin are distinguished by an extensive rough endopiasmic reticulum (RER) and a large Golgi complex (G). After culture on the substrate of laminin plus type IV collagen, myofilament bundles (F) are more prominent. L=lysosomes, M=mitochondria, N=nucleus. Bars=0.5 fim. expressed both in cultured arterial smooth muscle cells and in intimal lesions induced by balloon catheterization (unpublished observations). Other major components of the extracellular matrix may also be of importance in this context. The structure and function of smooth muscle cells cultured on or within a collagen gel was altered Moreover, the differen- tiated properties and growth of smooth muscle cells were changed after inhibition of proteoglycan synthesis by treatment with /3-D-xyloside.160 It has been suggested that heparin and heparan sulfate proteoglycans in the basement membrane of endothelial and smooth muscle cells inhibit the modulation of smooth muscle cells from a contractile to a synthetic phenotype in primary cul-

8 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 973 ture However, it is difficult to distinguish the effects on overall phenotypic properties and proliferation of the cells in these studies (see further below). We have not observed any distinct effect of heparin on the fine structural transformation of arterial smooth muscle cells in primary culture (unpublished observations). Role of Prostaglandins and Leukotrienes Numerous macrophages are found in atherosclerotic lesions These cells synthesize and release a large number of biologically potent molecules including growth factors, interleukins, hydrolytic enzymes, and adhesive glycoproteins They also represent a major source of prostaglandins and leukotrienes. These substances are formed from arachidonic acid via the cyclooxygenase and lipoxygenase pathways, respectively, and play important roles in allergic reactions and inflammation They are believed to exert their effects via specific receptors, and receptor antagonists are explored as potential therapeutic agents Considerable interest has been paid to the role of these substances in cardiovascular disease However, so far only a few studies have directed attention to their influence on the phenotypic state and proliferation of arterial smooth muscle cells. 182 Prostaglandin E, (PGE,), which shares many biological properties with prostaglandin l 2 (PGI 2 ), was found to accelerate the transition of rat aortic smooth muscle cells from a contractile to a synthetic phenotype early in primary culture as registered electron microscopically. 183 As a result, there was an earlier onset of serum-induced DNA synthesis and mitosis in PGE,-treated than in control cultures. However, once the cells had entered a synthetic phenotype, PGE, inhibited DNA synthesis and cell proliferation (see below). In addition, metabolites of PGI 2 were demonstrated to stimulate cholesteryl ester degradation in cultured arterial smooth muscle cells More recently, it was shown that leukotriene B 4 (LTB 4 ), C 4 (LTC 4 ), D 4 (LTD 4 ), and E 4 (LTE 4 ) all speed up the transition of rat aortic smooth muscle cells into a synthetic phenotype in primary culture. 186 Similar results were obtained with cells seeded on a plastic surface in serum-containing medium and cells seeded on a substrate of fibroneclin in serum-free medium. Furthermore, LTB 4, LTC 4, and LTD 4 stimulated the cells to enter the cell cycle earlier than in the controls (see below). These findings demonstrate that prostaglandins and leukotrienes are able to potently affect the differentiated state of arterial smooth muscle cells. The mechanisms of action are still unknown. Role of Polyamines The polyamines putrescine, spermidine, and spermine represent a group of ubiquitous, low molecular weight cations that are important in the control of cell differentiation and proliferation The major rate-limiting steps in the synthesis of polyamines are the reactions catalyzed by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). The two main substances used to block these enzymes are a-difluoromethylornithine (DFMO), a catalytic and irreversible inhibitor of ODC, and methylglyoxal-to/s(guanylhydrazone) (MGBG), a reversible and competitive inhibitor of SAMDC In a study on rat aortic smooth muscle cells, the modulation from a contractile to a synthetic phenotype was found to be associated with activation of polyamine metabolism. 190 ODC activity increased to a maximum after 2 days of culture and was accompanied by a successive rise in the cellular concentrations of putrescine, spermidine, and spermine. Treatment of the cultures with the polyamine synthesis inhibitors, DFMO and MGBG, distinctly slowed down the transition of the cells into a synthetic phenotype, as judged by following the fine structural reorganization of the cells and the onset of cellular proliferation. 190 As further discussed below, these drugs also inhibit the induction of DNA synthesis in subcultured smooth muscle cells by PDGF. 191 In addition, DFMO has been found to interfere with the proliferation and subsequent differentiation of myoblasts. 192 Remodulation from a Synthetic to a Contractile Phenotype In studies on rabbit and pig aortic smooth muscle, it was observed that the phenotypic modulation is reversible if the cells are seeded at a high initial density and a confluent monolayer is formed within less than 1 week of proliferation This was seen as an increase in the fractional volume occupied by myofilaments and in the expression of smooth muscle a-actin mrna as the cultures became confluent. 77 In studies on rat aortic smooth muscle cells cultured in the presence of serum, signs of reentry into a contractile phenotype at confluence have been more scarce However, preliminary experiments with subcultured cells detached with trypsin and reseeded in serum-free medium on substrates of fibronectin or laminin plus type IV collagen suggest that a partial remodulation may be possible (unpublished observations). On fibronectin, the percentage of cells with stress fibers stained for smooth muscle a-actin was low (Figure 4A), and the cells had a fine structure characteristic of a synthetic phenotype (Figure 5A). On laminin plus type IV collagen, there was an increase in the fraction of cells with smooth muscle a-actin positive stress fibers (Figure 4B), and distinct myofilament bundles reappeared in central parts of the cytoplasm (Figures 5B and 5C). These findings emphasize the importance of extracellular matrix components in the control of the differentiated properties of vascular smooth muscle cells (see above). In Vitro Cultivation as Experimental System to Study Regulation of Smooth Muscle Proliferation It is today widely recognized that smooth muscle cell proliferation is a major etiologic event in the formation of atherosclerotic lesions Since the early stages in the disease cannot be followed in humans, most in vivo studies have been made on naturally occurring or experimentally induced lesions in animals. 193 Autoradiography with tritiated thymidine was used to demonstrate that after mechanical injury of the endothelium, medial smooth muscle cells migrate into the intima and replicate However, it is difficult to study the regu-

9 974 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER 1990 Table 1. Induction of DNA Synthesis by Polypeptide Mitogens in Subcultures of Rat Aortic Smooth Muscle Cells Mitogen PDGF-AA PDGF-BB Basic FGF EGF IGF-I IL-1 TGF-/3 Fold stimulation Cmax (ng/ml) Lag phase (hours) Adult rat aortic smooth muscle cells (second or third passage) were grown to sub-confluence on glass coverslips, were incubated in serum-free medium for 48 hours, and were exposed to the indicated human recombinant polypeptide mitogens (basic FGF=bovine, recombinant) at different concentrations and for different times in the presence of tritiated thymidine. After fixation, the cultures were processed for autoradiography, and the percentage of labeled nuclei was determined. The results are presented as the approximate means of a series of experiments. C max =concentration where maximum effect is reached. Lag phase=time between mitogen addition and onset of DNA synthesis. PDGF=platelet-derived growth factor, FGF=fibroblast growth factor, EGF=epidermal growth factor, IGF-l=insulin-like growth factor-l, IL-1=interleukin-1, TGF-/3=transforming growth factor beta. lation of smooth muscle proliferation in vivo, and most of the literature in this field refers to in vitro systems. Induction of Cell Proliferation by Polypeptide Mitogens Like most other nontransformed cells, smooth muscle cells do not normally multiply in culture in the absence of serum. Ross and collaborators demonstrated that the active component in serum is of platelet origin. Similar observations were made on fibroblasts and glial cells Thereafter, a PDGF was purified and partially characterized The rapid growth in the knowledge on this mitogen during the following years has been outlined in recent reviews In short, PDGF is a cationic 30 kda protein composed of two polypeptide chains (A and B) that give rise to three disulfide-linked dimers (AA, AB, and BB). The A- and B-chains are encoded by separate but related genes, the B-chain gene being identical to the proto-oncogene c-s/s. 212 ' The different isoforms of PDGF exert their effects on target cells by interaction with high-affinity receptors of at least two types, the a-receptor, which binds either the A- or the B-chain, and the /3-receptor, which binds only the B-chain The binding of the ligand to the receptor gives rise to a complex biological response that ultimately leads to DNA replication and cell division Vascular smooth muscle cells were among the first cell types shown to have specific receptors for PDGF After binding to the receptors on the cell surface, PDGF is internalized by endocytosis and transferred to lysosomes for degradation In parallel, it was demonstrated that serum-starved smooth muscle cells enter the cell cycle, replicate their DNA, and divide in response to PDGF, 220 even under completely serum-free conditions Several studies have suggested an interaction between PDGF and insulin-like growth factor-l (IGF-I) in stimulating smooth muscle cell replication, and cells stimulated with PDGF were found to produce IGF-I However, IGF-I alone appears in most cases to be but a weak mitogen for vascular smooth muscle cells More recently, it has been demonstrated that vascular smooth muscle cells have both PDGF a- and /3-receptors and respond mitogenically to all isoforms of PDGF At least in subcultured smooth muscle cells, the a-receptors are less numerous than the /3-receptors and the mitogenic response to PDGF-AA is weaker than that to PDGF-AB and PDGF-BB. In addition to its effects on cellular replication, PDGF has been found to stimulate migration of smooth muscle CellS ,235 Other polypeptide growth factors of interest here are epidermal growth factor (EGF), and fibroblast growth factor (FGF) Vascular smooth muscle cells have receptors for and respond mitogenically to both of these factors Attention has also been paid to interleukin-1 (IL-1), and transforming growth factor-/? (TGF-/3) IL-1 was found to stimulate smooth muscle cell proliferation, apparently via induction of endogenous PDGF production. 252 With regard to TGF-/3, both inhibitory and stimulatory effects have been reported Recently, a growth-promoting effect of interleukin-6 (IL-6), a cytokine secreted by monocytes and macrophages, has also been reported. 258 Moreover, it was noted that smooth muscle cells stimulated with IL-1 or PDGF secrete large amounts of IL Our own observations on subcultured rat aortic smooth muscle cells have shown that PDGF-BB is the most potent growth factor, followed by FGF and EGF. On the other hand, PDGF-AA, IGF-I, IL-1, and TGF-/3 have relatively weak effects (Table 1). When vascular smooth muscle cells are grown in primary culture in a medium containing whole blood serum, the modulation into a synthetic phenotype is closely associated with the onset of cell proliferation In the presence of plasma-derived serum or under serum-free conditions, these processes can be studied separately Preliminary observations on rat aortic smooth muscle cells seeded on a substrate of fibronectin indicate that several growth factors are able to induce DNA synthesis and mitosis under these conditions (unpublished observations). In contrast to the situation in subcultured cells, an equipotent effect is obtained with PDGF-BB and PDGF-AA. 111 ' 112 ' 232 Thus, the mitogenic effect of different isoforms of PDGF may depend on the previous replicative history of the cells. FGF and EGF give less prominent responses, and with IGF-I, TGF-/3, and IL-1 no or only weak effects are seen (Table 2). Production of a Platelet-derived Growth Factor-like Mitogen In addition to being stimulated by exogenous mitogens, vascular smooth muscle cells have been found to synthesize and secrete growth-promoting agents on their own. First, it was demonstrated that aortic smooth muscle

10 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 975 Table 2. Induction of DNA Synthesis by Polypeptide Mitogens in Primary Cultures of Rat Aortic Smooth Muscle Cells Mitogen PDGF-AA PDGF-BB Basic FGF EGF IGF-I IL-1 TGF-/3 Fold stimulation Cmax (ng/ml) Lag phase (hours) Adult rat aortic smooth muscle cells (freshly isolated) were seeded on a substrate of fibronectin, were incubated in serumfree medium for 5 days, and were exposed to the indicated human recombinant polypeptide mitogens (basic FGF=bovine, recombinant) at different concentrations and for different times in the presence of tritiated thymidine. After fixation, the cultures were processed for autoradiography, and the percentage of labeled nuclei was determined. The results are presented as the approximate means of a series of experiments. C max =concentration where maximum effect is reached. Lag phase=time between mitogen addition and onset of DNA synthesis. See the legend for Table 1 for an explanation of the abbreviations. cells from young, but not from adult, rats release a PDGF-like molecule during successive passages in vitro. 260 In parallel, it was observed that adult rat aortic smooth muscle cells directly after the modulation into a synthetic phenotype and initiation of cell proliferation by an exogenous mitogen continue to proliferate in the absence of exogenous mitogen for several days. 70 Subsequently, it was shown that these cells release a PDGFlike molecule into the culture medium at several-fold higher levels than subcultured cells. 261 This was coupled to expression of mrna for the A-chain but not the B-chain of PDGF, raising the possibility that the mitogen produced by the cells is an A-chain homodimer. 262 Moreover, it was found that smooth muscle cells isolated from intimal lesions developing after balloon catheterization produced several-fold larger amounts of PDGF-like activity than cells isolated from the normal media. 263 It was then shown that aortic smooth muscle cells from newborn and adult rats both express increased levels of PDGF A-chain transcripts in vitro, whereas only cells from newborn rats express increased levels of PDGF B-chain transcripts. Further, cells from newborn rats secreted 60-fold more PDGF-like activity than cells from adult rats. 264 In another study, cell lysates were analyzed, and higher levels of PDGF-like activity were found in cells from old (>19 months) than from young (<3 months) rats. 265 It was also reported that both the PDGF A- and B-chain genes are expressed in rat aorta in vivo and that no major differences occur between normotensive and hypertensive animals Likewise, PDGF A- and B-chain transcripts were found in the normal human artery wall as well as in atherosclerotic lesions, and smooth muscle cells isolated from human atheroma express PDGF A-chain mrna and secrete a PDGF-like mitogen. 270 Human umbilical vein smooth muscle cells bind and respond to all isoforms of PDGF (AA<AB<BB) and secrete PDGF-AA-like material. 231 In partial contrast to these findings, cultured nonhuman primate (baboon) aortic smooth muscle cells were observed to transcribe both the PDGF A- and B-chain genes, but did not secrete detectable amounts of PDGF-like activity. 271 Using the primary culture system described above, with cells seeded on a substrate of fibronectin in serum-free medium, we have explored the production of PDGF-like material by rat aortic smooth muscle cells in some further detail. The results indicate that the expression of the PDGF A-chain gene increases as the cells go through the transition from a contractile to a synthetic phenotype. At the same time, PDGF receptor activity increases, but no detectable amounts of PDGF-like material are released into the medium, and the cells do not start to replicate until they have been exposed to exogenous PDGF. 272 Moreover, it was noted that the expression of PDGF a- and /3-receptor mrna increases in association with the phenotypic modulation, and that the cells specifically bind all isoforms of PDGF (AA<AB<BB). 232 Subsequent studies suggest that PDGF is the most potent inducer of endogenous PDGF production, although other growth factors may have a similar effect (unpublished observations); studies on passaged cells have indicated that angiotensin II and TGF-/3 stimulate the expression of PDGF A-chain mrna and secretion of PDGFlike molecules Several observations suggest that endogenous PDGF production is associated with autocrine or paracrine stimulation of cell replication. After the initial exposure to PDGF, there is a high rate of initiation of DNA synthesis in primary cultures for several days in the absence of exogenous stimuli, whereas a rapid return to baseline levels is seen in subcultures. 232 However, neither antibodies against PDGF nor suramin, a polyanionic drug that inhibits cellular binding of PDGF, 275 interferes with the initiation of DNA replication in the primary cultures. 276 These findings raise the possibility that the mitogen produced by the smooth muscle cells acts intracellularly, without prior release into the pericellular space. Production of Other Polypeptide Mitogens As mentioned earlier, cultured vascular smooth muscle cells produce IGF-I as part of the response to PDGF. 230 Human smooth muscle cells were shown to produce IL-1 when exposed to bacterial endotoxin or IL-1, Human and bovine smooth muscle cells have also been found to express and respond to acidic and basic FGF, respectively Moreover, after stimulation with several hormones and growth factors, human smooth muscle cells secrete the mitogenic peptide endothelin (see below). 279 Recently, a new growth factor distinct from PDGF and other previously described mitogens, was demonstrated to be produced by cultured rabbit smooth muscle cells after four passages or more Role of Vasoactive Hormones Studies on subcultured vascular smooth muscle cells have indicated that catecholamines like epinephrine and norepinephrine, potent vasoconstrictors in vivo, stimulate proliferation in the presence of serum and that the effect is mediated via oadrenergic receptors In a recent in

11 976 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER 1990 vivo study, it was further demonstrated that a-adrenergic agonists stimulate expression of the PDGF A-chain gene as well as other growth-related genes in rat aortic smooth muscle. 285 In this context, it is interesting to note that growth factors like PDGF and EGF have been found to induce contraction of rat aortic strips These findings suggest that the biological response of vascular smooth muscle to catecholamines and polypeptide mitogens depends on the phenotypic state of the cells; cells in a contractile phenotype are stimulated to contract and cells in a synthetic phenotype are stimulated to divide. Serotonin, a neurotransmitter and a substance released from degranulating platelets, was likewise observed to stimulate smooth muscle cell replication. 289 Angiotensin II, another potent vasoconstrictor, appears to be an exception from this rule. It binds to receptors on cultured vascular smooth muscle cells, and stimulates several steps in the signal transduction machinery for growth factors and hormones Nonetheless, angiotensin II has not been found to stimulate proliferation of vascular smooth muscle cells, neither alone nor in combination with serum or PDGF, but gives rise to an increased protein synthesis and cellular hypertrophy It has further been demonstrated that neuropeptides promote proliferation of vascular smooth muscle and other cells Substance P and substance K (neurokinin A) were shown to stimulate initiation of DNA synthesis in serum-starved rat aortic smooth muscle cells and human skin fibroblasts, and to add to the effect of a low concentration of PDGF. 299 In a similar manner, a growthpromoting effect of substance P was observed in an established line of embryonic rat aortic smooth muscle cells. 300 Subsequently, it was found that the response to substance K is dependent on the level of expression of the myc gene, possibly indicating that substance K alone is not able to induce all signals necessary for a mitogenic response. 301 Anyhow, substance P and substance K both stimulate formation of inositol phosphates in smooth muscle cells. 302 Recently, considerable interest has also been focused on endothelin, a potent vasoconstrictor peptide produced by vascular endothelial cells Endothelin binds specifically to human vascular smooth muscle cells in culture, and stimulates expression of the protooncogenes c-fos and c-myc as well as phospholipase C-mediated hydrolysis of phosphoinositides. 308 " 312 It was found to act as a mitogen for rat aortic smooth muscle cells, and to stimulate induction of DNA synthesis in mouse 3T3 fibroblasts Endothelin is thus another example of a substance with the dual function to stimulate vascular smooth muscle cells in a contractile phenotype to contract and cells in a synthetic phenotype to divide. Role of Prostaglandins and Leukotrienes During recent years, increasing interest has been directed to the possible role of prostaglandins and leukotrienes in the control of cell proliferation Several studies have indicated a growth-inhibitory effect of PGE, and PGE 2 on vascular smooth muscle cells grown in the presence of serum. 183 ' Similar results were obtained with PGI 2 and stable analogs of PGI In serum-starved cells, PGE, and PGE 2 inhibited induction of DNA synthesis by PDGF and this was associated with an increased intracellular concentration of cyclic AMP. 327 Adenosine and other agents that raise the cellular levels of cyclic AMP were likewise found to inhibit PDGFinduced DNA synthesis. 328 Cultured smooth muscle cells produce PGI 2 and other prostaglandins and the production is higher in growing than in resting cultures PDGF stimulates PGI 2 synthesis by bovine smooth muscle cells and mouse 3T3 fibroblasts and the effect is mediated via activation of phospholipase and several enzymes involved in prostaglandin biosynthesis Studies on cultured A10 smooth muscle cells revealed that EGF stimulates PGI 2 production if there is a concurrent stimulus that raises intracellular Ca 2+ levels. 340 In conclusion, these observations suggest that endogenous prostaglandins modulate the cellular response to PDGF and other growth factors. Studies in our laboratory have indicated that leukotrienes may also play an important role in the regulation of smooth muscle proliferation. As mentioned above, LTB 4, LTC 4, LTD,,, and LTE 4 all stimulate the conversion of rat aortic smooth muscle cells from a contractile to a synthetic phenotype early in primary culture. Moreover, LTB 4, LTC 4, and LTD 4 enhance the proliferative response to serum mitogens and, under serum-free conditions, induce DNA synthesis in newly modulated cells by themselves. 186 In addition, LTB 4, LTC 4, and LTD 4 induce DNA synthesis in subcultured rat aortic smooth muscle cells in the absence of other mitogens. 341 Treatment of the cells with indomethacin or acetylsalicylic acid, two cyclooxygenase inhibitors, blocked induction of DNA synthesis by LTB 4, suggesting that its effect is mediated by a cyclooxygenase product. Indirect support for this possibility was provided by the observation that the prereplicative lag phase is 6 to 8 hours longer with LTB 4 than with PDGF. The maximum effect of the leukotrienes was about half of that obtained with PDGF, and at suboptimal concentrations of PDGF, an additive effect of the leukotrienes was obtained. 341 Growth-promoting effects of leukotrienes have also been observed in cultures of glomerular epithelial cells, epidermal keratinocytes, 344 fibroblasts, 345 and endothelial cells. 346 In recent experiments, we have continued to examine the growth-stimulatory effect of LTB 4 on rat aortic smooth muscle cells, with particular emphasis on the role of endogenous cyclooxygenase products (unpublished observations). The results reveal that arachidonic acid, PGF 2a, PGE 2, and PGI 2 all are able to induce DNA synthesis under serum-free conditions and in the absence of other mitogens. When given together with PDGF, PGE 2 and PGI 2 showed an inhibitory effect, whereas PGF 2a lacked any distinct effect. DNA synthesis could also be induced with hydroxyeicosatetraenoic acids (HETEs), products derived from arachidonic acid primarily via the lipoxygenase pathway. 347 The most prominent effect was seen with 15-HETE, which in rat aortic smooth muscle cells can be produced via the cyclooxygenase pathway. 348 Radioimmunologic analyses indicated that the smooth muscle cells are able to produce PGF 2m PGE 2, PGI 2, and 15-HETE and that the

12 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 977 CONTRACTILE PHENOTYPE I SYNTHETIC PHENOTYPE I i fibronectin-mediated change in differentiated properties stimulation of cell proliferation by exogenous PDGF stimulation of continued cell proliferation by endogenous PDGF PDGF Figure 6. Schematic model of the role of fibronectin and platelet-derived growth factor (PDGF) in the regulation of differentiated properties and proliferation of arterial smooth muscle cells. production is blocked by indomethacin. Moreover, induction of DNA synthesis by conditioned media from smooth muscle cells exposed to LTB 4 was inhibited by antibodies against PDGF (unpublished observations). Taken together, these findings suggest that the growth-promoting effect of LTB 4 may be mediated coordinately by prostaglandins, HETEs, and PDGF produced by the smooth muscle cells themselves. In studies on murine smooth muscle cells, treatment with LTD 4 was found to be associated with increased phospholipase A 2 activity and production of prostaglandins. 349 Role of Polyamines As mentioned above, it has long been recognized that polyamines play a crucial role in the control of cell differentiation and proliferation However, few studies have dealt with the effects of pure growth factors on polyamine metabolism in cultured cells. In an investigation on rat aortic smooth muscle cells, we found that PDGF causes an increase in ODC activity and a rise in the cellular content of putrescine. 191 The inhibitor DFMO blocked the increase in ODC activity as well as the accumulation of putrescine. Moreover, DFMO inhibited induction of DNA synthesis by PDGF without affecting the length of the prereplicative lag phase. This effect was counteracted by the addition of polyamines to the culture medium, and exogenous polyamines were found to stimulate the initiation of DNA synthesis in PDGF-free medium (spermine>spermidine>putrescine). On the basis of these findings, it is concluded that induction of ODC and putrescine synthesis are integral parts in the mitogenic response of smooth muscle cells to PDGF. 191 In parallel, it was observed that exposure of quiescent mouse 3T3 fibroblasts to PDGF induced ODC activity and that treatment of the cells with DFMO inhibited the effect of the mitogen on DNA synthesis S2 Other growth factors, like FGF and EGF, have also been found to induce ODC activity in fibroblastic cells Role of Cytoskeleton Microtubules and actin filaments are the two major components of the cytoskeleton in mammalian cells and fulfill important functions in the control of cell shape, motility, and intracellular transport. Furthermore, these structures play crucial roles during cell division; microtubules build up the mitotic spindle that separates the chromosomes, 354 and actin filaments take an active part in the division of the cytoplasm during cytokinesis. 355 Distinct changes in the organization of the cytoskeleton have been observed in cells stimulated with growth factors. EGF caused actin reorganization and membrane ruffling in responsive cells Similar findings were subsequently made with PDGF, and the response appeared to be mediated via the /3-receptor. 362 Studies on vascular smooth muscle cells have indicated that PDGF, but not EGF or IGF-I, causes a rapid and reversible disappearance of the protein vinculin from adhesion plaques as well as a disruption of actin filament bundles. 363 These changes may be important for the stimulation of cell motility and cell division, which both require detachment of the cells from the substrate. With regard to microtubules, clearcut alterations in intracellular organization have been more difficult to demonstrate in connection with the early stages of growth factor stimulation. Nevertheless, a large number of studies indicate that treatment of cells with microtubuledisruptive drugs like colchicine or colcemid either potentiate or inhibit the effect of growth factors on initiation of DNA synthesis, or in some cases even stimulate initiation of DNA synthesis on their own In a study on rat aortic smooth muscle cells, a stimulatory effect of colchicine on induction of DNA synthesis was observed in serum-free medium and in the presence of plasmaderived serum. On the other hand, a weak inhibitory effect was obtained in cells exposed to PDGF or whole blood serum. 368 These findings suggest that partial microtubule disassembly may be an inherent step in the processes that lead the cells through the G, and into the S phase of the cell cycle. Role of Extracellular Matrix Components Numerous investigations have emphasized the role of extracellular matrix components in the control of vascular smooth muscle proliferation in culture. An extracellular matrix produced by endothelial cells was found to promote the growth and increase the life span of bovine aortic smooth muscle cells. 241 Subsequently, it was observed that heparan sulfate produced by endothelial cells or smooth muscle cells, as well as heparin, inhibits smooth muscle cell proliferation Heparin was likewise found to suppress in vivo proliferation of smooth

13 978 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER 1990 muscle cells in injuried arteries Structural studies indicated that heparin's antiproliferative activity depends on oligosaccharide size and charge, but not on anticoagulant activity Heparin binds to cells and is ingested by endocytosis It affects several steps in the transition of the cells from the G o to the S phase of the cell cycle and may have multiple targets for its antiproliferative effect Heparin has been reported to modulate the secretion of major proteins by cultured smooth muscle cells, including two 35 to 40 kda proteins, a 60 kda collagen, and thrombospondin In addition, a 78 kda heparin-binding protein involved in the inhibition of smooth muscle cell proliferation was described. 392 Stimulation of smooth muscle cells with PDGF was found to induce synthesis and secretion of thrombospondin, an extracellular matrix glycoprotein that acts synergistically with EGF to promote entrance into the S phase of the cell cycle; heparin inhibited the effect of thrombospondin on EGF-induced mitogenesis as well as the incorporation of thrombospondin in the extracellular matrix The effect of heparin on smooth muscle cell proliferation does not appear to be mediated solely by decreased availability or activity of PDGF. 396 The antiproliferative effect of heparin can be reversed by EGF, 397 and heparin and heparin-like molecules were found to regulate the number of EGF receptors on vascular smooth muscle cells. 398 It was also noted that cultured smooth muscle cells from spontaneously hypertensive rats are less susceptible than are cells from control rats to growth inhibition by heparin. 399 Role of Protein Phosphorylation, Phosphatidylinositol Turnover, and Ion Fluxes The binding of PDGF and other polypeptide mitogens to their receptors elicits a cascade of biological responses, 400 including protein phosphorylation, phosphatidylinositol turnover, ion fluxes, and increased expression of specific genes It is still unclear exactly how these reactions relate to each other and to the final result of the stimulation, i.e., DNA synthesis and mitosis. In any case, they represent possible targets through which the various factors discussed above may act to influence cell proliferation. The PDGF and EGF receptors are protein kinases that phosphorylate themselves and a number of other cellular proteins on tyrosine residues. Recent studies on mutated receptors have indicated that this activity is required to evoke a full mitogenic response The phosphate groups added to the proteins are in most cases rapidly removed by a group of enzymes referred to as protein tyrosine phosphatases Since cells must be exposed to the growth factors for prolonged periods of time in order to enter the S phase, it is possible that the receptors have to be activated more than once to be effective. In this context, it is interesting to note that ions of vanadium, an inhibitor of protein tyrosine phosphatases, stimulate growth of fibroblastic cells In studies on rat aortic smooth muscle cells, we have observed similar effects. Addition of vanadium ions (10 /im) to serum-starved cultures induced DNA synthesis in a smaller fraction of the cells. Moreover, the response to low concentrations of PDGF (<5 ng/ml) and to short exposures to PDGF (<10 hours) was distinctly enhanced by vanadium ions (unpublished observations). Although other mechanisms of action cannot be excluded, these findings support the idea that protein tyrosine phosphorylation is an important step in the stimulation of smooth muscle cell proliferation. Growth factors and other agonists bring about hydrolysis of phospoinositides (minor phospolipids present in all eukaryotic cells) by phospholipase C. This gives rise to two important second messengers: inositol trisphosphate that mobilizes calcium ions from intracellular storage sites and diacylglycerol that activates protein kinase C, an enzyme that phosphorylates proteins on serine and threonine residues A possible mechanism of action was revealed when PDGF was found to induce an association of phospholipase C-y with the PDGF receptor complex as well as tyrosine phosphorylation of the enzyme However, studies on vascular smooth muscle cells have indicated that phosphoinositide breakdown is neither sufficient nor necessary for a mitogenic response. Both mitogenic and nonmitogenic agonists stimulate phosphoinositide hydrolysis ' Moreover, there is not always a good correlation between the growth-promoting effect of PDGF and its effect on phosphoinositide hydrolysis In any case, smooth muscle cell proliferation is to some extent dependent on the calcium ion concentration of the extracellular medium, 428 and calcium antagonists have been found to inhibit induction of DNA synthesis by PDGF as well as PDGFinduced degradation of phosphoinositides Interestingly, it was recently observed that PDGF stimulates the association of phosphatidylinositol-3'-kinase with the PDGF receptor complex and that this leads to production of novel phosphoinositides, which may take part in mediating the mitogenic effect of PDGF Activation of cell proliferation is also associated with Na + /H + exchange over the plasma membrane and a temporary alkalinization of the cytoplasm, but the functional significance of this event remains unclear In vascular smooth muscle cells, agents with, as well as without, growth-promoting effect have been shown to activate Na + /H + exchange, leading to a biphasic shift in cytoplasmic ph As previously reported for fibroblastic cells, amiloride (an inhibitor of the Na + /H + exchanger) was found to potently block induction of DNA synthesis by PDGF or serum in rat aortic smooth muscle cells (unpublished observations). Therefore, it seems likely that Na7H + exchange and cytoplasmic alkalinization play at least a permissive role in smooth muscle proliferation. Perspectives and Summary In the adult organism, arterial smooth muscle cells are in a highly differentiated, contractile phenotype. However, they have the potential to revert to a synthetic phenotype, similar to that prevailing in the embryo and the young growing organism. This leads to the cells losing their contractility and instead gaining the ability to secrete extracellular matrix components and divide, and this is an important early event in atherogenesis. Based on the ex-

14 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al. 979 Table 3. Overview of Substances Reported to Stimulate Proliferation of Vascular Smooth Muscle Cells in Culture Substance References Polypeptide mitogens Platelet-derived growth factor (PDGF) 69, 70, 111, 112, 220, 221, , 231, 232 Insulin-like growth factor-l (IGF-I) Epidermal growth factor (EGF) Fibroblast growth factor (FGF) 241, 244, 245 lnterleukin-1 (IL-1) lnterleukin-6 (IL-6) 258 Transforming growth factor-/3 (TGF-0) , 274 Smooth muscle cell derived growth factor (SDGF) 280, 281 Vasoactive hormones Epinephrine/norepinephrine Serotonin 289 Substance P 299, 300, 302 Substance K (neurokinin A) 299, 301, 302 Endothelin 309, 310, 312 Prostaglandins PGF 2(, (see text) PGE 2 (see text) PGI 2 (see text) Leukotrienes LTB 4 186, 341 LTC 4 186, 341 LTD 4 186,341 perimental data presented in this review, we propose a simplified model for the regulation of differentiated properties and proliferation of arterial smooth muscle cells (Figure 6). The basement membrane, a sheet-like network made up of laminin, type IV collagen, and heparan sulfate proteoglycans, normally helps to keep the cells in a contractile phenotype. If this barrier is damaged, the plasma membrane may interact with other macromolecules derived from the blood or produced locally by the cells in the vessel wall. Adhesive glycoproteins such as fibronectin bind to integrin receptors on the cell surface and induce a transition of the cells from a contractile to a synthetic phenotype. This process includes loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex, organelles which take part in the synthesis and secretion of extracellular matrix components. In parallel, there is a reduced expression of o-actin mrna, and the cells start to express PDGF a- and /3-receptors as well as mrna for the A-chain of PDGF. In response to stimulation with exogenous PDGF (AA, AB, or BB), the cells replicate their DNA and divide. Moreover, they start to produce PDGF (AA) on their own, which helps them to continue to proliferate in the absence of exogenous mitogen for a limited number of generations. Growth-promoting agents may be derived from the blood, degranulating platelets, leukocytes invading the vessel wall, or endothelial cells. Among these agents, polypeptide mitogens, vasoactive hormones, prostaglandins, and leukotrienes are the most important (Table 3). This simplified model can be used as a framework for future studies on the regulation of differentiated properties and proliferation of arterial smooth muscle cells during in vitro cultivation as well as during atherogenesis. It also suggests possible ways in which to interfere pharmacologically with the phenotypic modulation and the subsequent proliferation of the smooth muscle cells. References 1. Evans HM. On the development of the aortae, cardinal and umbilical veins, and the other blood vessels of vertebrate embryos from capillaries. Anat Rec 1909:3: Pardanaud L, Altmann C, Kitos P, Dieterlen-Llevre F, Buck CA. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 1987; 100: Pallie W. Embryology of the human arterial system (arteriogenesis). In: Schwartz CJ, Werthessen NT, Wolf S, eds. Structure and function of the circulation, vol 1. New York: Plenum Press, 1980: Gerrity RG, Cliff WJ. The aortic tunica media of the developing rat. I. Quantitative stereologic and biochemical analysis. Lab Invest 1975:32: Nakamura H. Electron microscopic study of the prenatal development of the thoracic aorta in the rat. Am J Anat 1988;181: Ross R, Klebanoff SJ. The smooth muscle cell. I. In vivo synthesis of connnective tissue proteins. J Cell Biol 1971; 50: Gerrity RG, Adams EP, Cliff WJ. The aortic tunica media of the developing rat. II. Incorporation by medial cells of 3 H-proline into collagen and elastin: autoradiographic and chemical studies. Lab Invest 1975;32: Davidson JM, Hill KE, Alford JL. Developmental changes in collagen and elastin biosynthesis in the porcine aorta. Dev Biol 1986;118:

15 980 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER Kocher O, Skalli O, Cerutti D, Gabbiani F, Gabbiani G. Cytoskeletal features of rat aortic cells during development. An electron microscopic, immunohistochemical, and biochemical study. Circ Res 1985:56: Kocher O, Gabbiani G. Expression of actin mrna in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture. Differentiation 1986; 32: Owens GK, Thompson MM. Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation. J Biol Chem 1986;261: Nikkari ST, Rantala I, Pystynen P, Nikkari T. Characterization of the phenotype of smooth muscle cells in human fetal aorta on the basis of ultrastructure, immunofluorescence, and the composition of cytoskeletal and cytocontractile proteins. Atherosclerosis 1988;74: Gabbiani G, Schmid E, Winter S, et al. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific a-type actin. Proc Natl Acad Sci USA 1981;78: Berner PF, Frank E, Holtzer H, Somlyo AP. The intermediate filament proteins of rabbit vascular smooth muscle: immunofluorescent studies of desmin and vimentin. J Muscle Res Cell Motility 1981;2: Schmid E, Osborn M, Rungger-Brandle E, Gabbiani G, Weber K, Franke WW. Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta. Exp Cell Res 1982:137: Fujimoto T, Tokuyasu KT, Singer SJ. Direct morphological demonstration of the coexistence of vimentin and desmin in the same intermediate filaments of vascular smooth muscle cells. J Submicrosc Cytol Pathol 1987; 19: Larson DM, Fujiwara K, Alexander RW, Gimbrone MA Jr. Heterogeneity of myosin antigenic expression in vascular smooth muscle cells in vivo. Lab Invest 1984;50: Longtine JA, Pinkus GS, Fujiwara K, Corson JM. Immunohistochemical localization of smooth muscle myosin in normal human tissues. J Histochem Cytochem 1985;33: Rhodin JAG. Architecture of the vessel wall. In: Bohr DF, Somlyo AP, Sparks HV Jr, eds. Handbook of physiology, the cardiovascular system, vascular smooth muscle (vol II). Bethesda: Am Physiol Soc, 1980: Somlyo AV. Ultrastructure of vascular smooth muscle. In: Bohr DF, Somlyo AP, Sparks HV Jr, eds. Handbook of physiology, the cardiovascular system, vascular smooth muscle (vol II). Bethesda: Am Physiol Soc, 1980: Timpl R, Dziadek M. Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol 1986;29: Timpl R. Structure and biological activity of basement membrane proteins. Eur J Biochem 1989; 180: Inoue S. Ultrastructure of basement membranes. Int Rev Cytol 1989; 117: Heickendorff L. The basement membrane of arterial smooth muscle cells. Acta Pathol Microbiol Immunol Scand 1989;97(suppl 9): Sandberg LB, Soskel NT, Leslie JG. Elastin structure, biosynthesis, and relation to disease states. N Engl J Med 1981;304: Rosenbloom J. Elastin: an overview. Methods Enzymol 1987; 144: Barnes MJ. Collagens in atherosclerosis. Coll Relat Res 1985:5: Mayne R. Collagenous proteins of blood vessels. Arteriosclerosis 1986:6: Poole AR. Proteoglycans in health and disease: structures and functions. Biochem J 1986:236: Wight TN. Cell biology of arterial proteoglycans. Arteriosclerosis 1989;9: Pearson ML, Epstein HF. Muscle development: molecular and cellular control. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982: Campbell GR, Chamley-Campbell JH. The cellular pathobiology of atherosclerosis. Pathology 1981;13: Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 1979,59: Ross R. The pathogenesis of atherosclerosis an update. N Engl J Med 1986;314: Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res 1986; 58: Munro JM, Cotran RS. The pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest 1988:58: Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells. Am J Physiol 1989;257:H1755-H Watanabe T, Hirata M, Yoshlkawa Y, Nagafuchi Y, Toyoshima H, Watanabe T. Role of macrophages in atherosclerosis. Sequential observations of cholesterolinduced rabbit aortic lesion by the immunoperoxidase technique using monoclonal antimacrophage antibody. Lab Invest 1985:53: Aqel NM, Ball RY, Waldmann H, Mitchinson MJ. Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies. J Pathol 1985; 146: Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6: Gown AM, Tsukada T, Ross R. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol 1986; 125: Tsukada T, Rosenfeld M, Ross R, Gown AM. Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis 1986;6: Ross R, Wight TN, Strandness E, Thiele B. Human atherosclerosis. I. Cell constitution and characteristics of advanced lesions of the superficial femoral artery. Am J Pathol 1984; 114: Mosse PRL, Campbell GR, Wang ZL, Campbell JH. Smooth muscle phenotypic expression in human carotid arteries. I. Comparison of cells from diffuse intimal thickenings adjacent to atheromatous plaques with those of the media. Lab Invest 1985:53: Mosse PRL, Campbell GR, Campbell JH. Smooth muscle phenotypic expression in human carotid arteries. II. Atherosclerosis-free diffuse intimal thickenings compared with the media. Arteriosclerosis 1986:6: Grunwald J, Fingerle J, Hammerle H, Betz E, Haudenschild CC. Cytocontractile structures and proteins of smooth muscle cells during the formation of experimental lesions. Exp Mol Pathol 1987;46: Manderson JA, Mosse PRL, Safstrom JA, Young SB, Campbell GR. Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype. Arteriosclerosis 1989; Manderson JA, Cocks TM, Campbell GR. Balloon catheter injury to rabbit carotid artery. II. Selective increase in reactivity to some vasoconstrictor drugs. Arteriosclerosis 1989;9: Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J, Weber K. Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest 1984;73: Kocher O, Skalli O, Bloom WS, Gabbiani G. Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and experimental intimal thickening. Lab Invest 1984;50:

16 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al Kocher O, Gabblani G. Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells. Hum Pathol 1986; 17: Barja F, Coughlin C, Belln D, Gabblani G. Actin isoform synthesis and mrna levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro. Lab Invest 1986;55: Clowes AW, Clowes MM, Kocher O, Ropraz P, Chaponnler C, Gabblani G. Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin. J Cell Biol 1988:107: Benzonana G, Skalll O, Gabblani G. Correlation between the distribution of smooth muscle or non muscle myosins and a-smooth muscle actin in normal and pathological soft tissues. Cell Motil Cytoskeleton 1988:11: Gabblani G, Rungger-Brandle E, De Chastonay C, Franke WW. Vimentin-containing smooth muscle cells in aortic intimal thickening after endothelial injury. Lab Invest 1982:47: Osborn M, Caselitz J, Piischel K, Weber K. Intermediate filament expression in human vascular smooth muscle and in arteriosclerotic plaques. Virchows Arch [A] 1987;411: Jahn L, Franke WW. High frequency of cytokeratinproducing smooth muscle cells in human atherosclerotic plaques. Differentiation 1989;40: Jonasson L, Holm J, Skalll O, Gabbianl G, Hansson GK. Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis. J Clin Invest 1985:76: Hansson GK, Jonasson L, Holm J, Claesson-Welsh L. Class II MHC antigen expression in the atherosclerotic plaque: smooth muscle cells express HLA-DR, HLA-DQ, and the invariant gamma chain. Clin Exp Immunol 1986; 64: Jonasson L, Holm J, Hansson GK. Smooth muscle cells express la antigens during arterial response to injury. Lab Invest 1988:58: Lamazlere J-MD, Desmoullere A, Pascal M, Larrue J. Detection of atherosclerotic plaque with two monoclonal antibodies. 2P1A2 monoclonal antibody is specific for smooth muscle cells in atherosclerotic plaque. Atherosclerosis 1988:74: Prlntseva OJ, Peclo MM, Tjurmln AV, et al. A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions. Am J Pathol 1989:134: Murray MR, Kopech G. A bibliography of the research in tissue culture 1894/1950. New York: Academic Press, 1953: Murray MR. Muscle. In: Willmer EN, ed. Cells and tissues in culture. Methods, biology and physiology, vol 2. London: Academic Press, 1965: Campbell JH, Campbell GR. Vascular smooth muscle in culture. Boca Raton: CRC Press, 1987: Piper HM. Cell culture techniques in heart and vessel research. Berlin: Springer-Verlag, 1990: Fritz KE, Jarmolych J, Daoud AS. Association of DNA synthesis and apparent dedifferentiation of aortic smooth muscle cells in vitro. Exp Mol Pathol 1970;12: Chamley JH, Campbell GR, McConnell JD, Groschel- Stewart U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res 1977; 177: Chamley-Campbell JH, Campbell GR, Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J Cell Biol 1981;89: Thyberg J, Palmberg L, Nllsson J. Kslazek T, Sjolund M. Phenotype modulation in primary cultures of arterial smooth muscle cells: on the role of platelet-derived growth factor. Differentiation 1983:25: Thyberg J, Nllsson J, Palmberg L, Sjolund M. Adult human arterial smooth muscle cells in primary culture: modulation from contractile to synthetic phenotype. Cell Tissue Res 1985;239: Palmberg L, Sjolund M, Thyberg J. Phenotype modulation in primary cultures of arterial smooth-muscle cells: reorganization of the cytoskeleton and activation of synthetic activities. Differentiation 1985;29: Gown AM, Vogel AM, Gordon D, Lu PL. A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. J Cell Biol 1985:100: Skalll O, Bloom WS, Ropraz P, Azzarone B, Gabbianl G. Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations. J Submicrosc Cytol Pathol 1986; 18: Owens GK, Loeb A, Gordon D, Thompson MM. Expression of smooth muscle-specific a-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation. J Cell Biol 1986; 102: Skalll O, Ropraz P, Trzeclak A, Benzonana G, Glllessen D, Gabbianl G. A monoclonal antibody against a-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986:103: Campbell JH, Kocher O, Skalll O, Gabbianl G, Campbell GR. Cytodifferentiation and expression of a-smooth muscle actin mrna and protein during primary culture of aortic smooth muscle cells: correlation with cell density and proliferative state. Arteriosclerosis 1989:9: Kocher O, Gabblani G. Analysis of o-smooth-muscle actin mrna expression in rat aortic smooth-muscle cells using a specific cdna probe. Differentiation 1987;34: Blank RS, Thompson MM, Owens GK. Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells. J Cell Biol 1988:107: Corjay MH, Thompson MM, Lynch KR, Owens GK. Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle a-actin and nonmuscle /3-actin mrna expression in cultured rat aortic smooth muscle cells. J Biol Chem 1989:264: Absher M, Woodcock-Mitchell J, Mitchell J, Baldor L, Low R, Warshaw D. Characterization of vascular smooth muscle cell phenotype in long-term culture. In Vitro Cell DevBiol 1989;25: Liau G, Janat MF, Wirth PJ. Regulation of a-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells. J Cell Physiol 1990;142: Fager G, Hansson GK, Gown AM, Larson DM, Skalll O, Bondjers G. Human arterial smooth cells in culture: inverse relationship between proliferation and expression of contractile proteins. In Vitro Cell Dev Biol 1989;25: Dartsch PC, Voisard R, Baurledel G, Hofling B, Betz E. Growth characteristics and cytoskeletal organization of cultured smooth muscle cells from human primary stenosing and restenosing lesions. Arteriosclerosis 1990; 10: Dartsch PC, Weiss H-D, Betz E. Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements. Eur J Cell Biol 1990:51: Franke WW, Schmid E, Vandekerckhove J, Weber K. A permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type. J Cell Biol 1980:87: Strauch AR, Rubenstein PA. Induction of vascular smooth muscle a-isoactin expression in BC 3 H1 cells. J Biol Chem 1984;259: Strauch AR, Rubenstein PA. A vascular smooth muscle a-isoactin biosynthetic intermediate in BC 3 H1 cells: identification of acetylcysteine at the NH 2 terminus. J Biol Chem 1984;259:

17 982 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER Strauch AR, Offord JD, Chalkley R, Rubenstein PA. Characterization of actin mrna levels during BC 3 H1 cell differentiation. J Biol Chem 1986;261: Strauch AR, Reeser JC. Sequential expression of smooth muscle and sarcomeric a-actin isoforms during BC3H1 cell differentiation. J Biol Chem 1989;264: Nachtigal M, Nagpal ML, Greenspan P, Nachtigal SA, Legrand A. Characterization of a continuous smooth muscle cell line derived from rabbit aorta. In Vitro Cell Dev Biol 1989;25: Reilly CF. Rat vascular smooth muscle cells immortalized with SV40 large T antigen possess defined smooth muscle cell characteristics including growth inhibition by heparin. J Cell Physiol 1990:142: Groschel-Stewart U, Chamley JH, Campbell GR, Bumstock G. Changes in myosin distribution in dedifferentiating and ^differentiating smooth muscle cells in tissue culture. Cell Tissue Res 1975; 165: Larson DM, Fujiwara K, Alexander RW, Gimbrone MA Jr. Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression. J Cell Biol 1984;99: Rovner AS, Murphy RA, Owens GK. Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. J Biol Chem 1986; 261: Kawamoto S, Adelstein RS. Characterization of myosin heavy chains in cultured aorta smooth muscle cells: a comparative study. J Biol Chem 1987;262: Hammerle H, Fingerle J, Rupp J, Griinwald J, Betz E, Haudenschild CC. Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells. Exp Cell Res 1988; 178: Chamley-Campbell J, Campbell GR, Groschel-Stewart U, Burnstock G. FITC-labelled antibody staining of tropomyosin-containing fibrils in smooth, cardiac and skeletal muscle cells, prefusion myoblasts, fibroblasts, endothelial cells and 3T3 cells in culture. Cell Tissue Res 1977; 183: Travo P, Weber K, Osborn M. Co-existence of vimentin and desmin type intermediate filaments in a subpopulation of adult rat vascular smooth muscle cells growing in primary culture. Exp Cell Res 1982; 139: Sjolund M, Madsen K, von der Mark K, Thyberg J. Phenotype modulation in primary cultures of smoothmuscle cells from rat aorta: synthesis of collagen and elastin. Differentiation 1986;32: Ang AH, Tachas G, Campbell JH, Bateman JF, Campbell GR. Collagen synthesis by cultured rabbit aortic smooth-muscle cells: alteration with phenotype. Biochem J 1990;265: Merrilees MJ, Campbell JH, Spanldis E, Campbell GR. Glycosaminoglycan synthesis by smooth muscle cells of differing phenotype and their response to endothelial cell conditioned medium. Atherosclerosis 1990;81: Hinek A, Thyberg J. Electron microscopic observations on the formation of elastic fibers in primary cultures of aortic smooth muscle cells. J Ultrastruct Res 1977;60: Oakes BW, Batty AC, Handley CJ, Sandberg LB. The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study. Eur J Cell Biol 1982;27: Yamamoto H, Kanaide H, Nakamura M. Metabolism of glycosaminoglycans of cultured rat aortic smooth muscle cells altered during subculture. Br J Exp Pathol 1983;64: Barone LM, Wolfe BL, Faris B, Franzblau C. Elastin mrna levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures. Biochemistry 1988;27: Burke JM, Ross R. Synthesis of connective tissue macromolecules by smooth muscle. Int Rev Connect Tissue Res 1979:8: Campbell JH, Popadynec L, Nestel PJ, Campbell GR. Lipid accumulation in arterial smooth muscle cells: influence of phenotype. Atherosclerosis 1983;47: Campbell JH, Reardon MF, Campbell GR, Nestel PJ. Metabolism of atherogenic lipoproteins by smooth muscle cells of different phenotype in culture. Arteriosclerosis 1985;5: Parlavecchia M, Skalll O, Gabbiani G. LDL accumulation in cultured rat aortic smooth muscle cells with different cytoskeletal phenotypes. J Vascular Med Biol 1989;1: Hedin U, Thyberg J. Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype. Differentiation 1987;33: Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol 1988;107: Ruoslahti E, Hayman EG, Pierschbacher MD. Extracellular matrices and cell adhesion. Arteriosclerosis 1985;5: Hynes RO. Fibronectins. Sci Am 1986;254: Akiyama SK, Yamada KM. Fibronectin. Adv Enzymol Related Areas Mol Biol 1987;59: Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem 1988;57: Holderbaum D, Ehrhart LA. Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro. J Cell Physiol 1986;126: Hynes RO. Integrins: a family of cell surface receptors. Cell 1987;48: Buck CA, Horwitz AF. Cell surface receptors for extracellular matrix molecules. Annu Rev Cell Biol 1987,3: McDonald JA. Receptors for extracellular matrix components. Am J Physiol 1989;257:L331-L Akiyama SK, Nagata K, Yamada KM. Cell surface receptors for extracellular matrix components. Biochim Biophys Acta 1990;1031: Hedin U, Bottger BA, Luthman J, Johansson S, Thyberg J. A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype. Dev Biol 1989;133: Bottger BA, Hedin U, Johansson S, Thyberg J. Integrintype fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties. Differentiation 1989;41: Hedin U, Sjolund M, Hultgardh-Nilsson A, Thyberg J. Changes in expression and organization of smooth muscle-specific a-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype. Differentiation (in press) 125. Risau W, Lemmon V. Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev Biol 1988; 125: Stenman S, von Smitten K, Vaheri A. Fibronectin and atherosclerosis. Acta Med Scand 1980;Suppl 642: Orekhov AN, Andreeva ER, Shekhonin BV, Tertov W, Smirnov VN. Content and localization of fibronectin in normal intima, atherosclerotic plaque, and underlying media of human aorta. Arteriosclerosis 1984;53: Smith EB, Ashall C. Fibronectin distribution in human aortic intima and atherosclerotic lesions: concentration of soluble and collagenase-releasable fractions. Biochim Biophys Acta 1986;880: Voss B, Rauterberg J. Localization of collagen types I, III, IV and V, fibronectin and laminin in human arteries by the indirect immunofluorescence method. Pathol Res Pract 1986; 181:

18 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al Shekhonin BV, Domogatsky SP, Idelson GL, Koteliansky VE, Rukosuev VS. Relative distribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intima of human arteries. Atherosclerosis 1987;67: Glukhova MA, Frld MG, Shekhonin BV, et al. Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J Cell Biol 1989; 109: Podleskl TR, Greenberg I, Schlesslnger J, Yamada KM. Fibronectin delays the fusion of U myoblasts. Exp Cell Res 1979;122: Kuhl U, Ocalan M, Tlmpl R, von der Mark K. Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro. Dev Biol 1986:117: von der Mark K, Ocalan M. Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 1989:40: Chen LB. Alterations in cell surface LETS protein during myogenesis. Cell 1977:10: Furcht LT, Mosher DF, Wendelschafer-Crabb G. Immunocytochemical localization of fibronectin (LETS protein) on the surface of L6 myoblasts: light and electron microscopic studies. Cell 1978; 13: Kuhl U, Timpl R, von der Mark K. Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes. Dev Biol 1982:93: Meola G, Scarplnl E, Vellcogna M, et al. Analysis of fibronectin expression during human muscle differentiation. Basic Appl Histochem 1986:30: Chung CY, Kang M-S. Correlation between fibronectin and its receptor in chick myoblast differentiation. J Cell Physiol 1990:142: Ben-Ze'ev A. Cell shape, the complex cellular networks, and gene expression: cytoskeletal protein genes as a model system. In: Strohman RC, Wolf S, eds. Gene expression in muscle. New York: Plenum Publishing Corp., 1985; Watt FM. The extracellular matrix and cell shape. Trends Biochem Sci 1986; 11: Blssell MJ, Barcellos-Hoff MH. The influence of extracellular matrix on gene expression: is structure the message? J Cell Sci 1987;Suppl 8: Herman IM. Extracellular matrix-cytoskeletal interactions in vascular cells. Tissue Cell 1987; 19: Thyberg J, Hedin U, Bottger BA. Attachment substrates for smooth muscle cells. In: Piper HM, ed. Cell culture techniques in heart and vessel research. Berlin: Springer- Verlag, 1990: Ruoslahtl E, Suzuki S, Hayman EG, III CR, Plerschbacher MD. Purification and characterization of vitronectin. Methods Enzymol 1987; 144: Izuml M, Yamada KM, Hayashl M. Vitronectin exists in two structurally and functionally distinct forms in human plasma. Biochim Biophys Acta 1989:990: Pytela R, Plerschbacher MD, Ruoslahtl E. A 125/115- kda cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc Natl Acad Sci USA 1985;82: Lawler J. The structural and functional properties of thrombospondin. Blood 1986:67: Sllversteln RL, Leung LLK, Nachman RL. Thrombospondin: a versatile multifunctional glycoprotein. Arteriosclerosis 1986:6: Frazler WA. Thrombospondin: a modular adhesive glycoprotein of platelets and nucleated cells. J Cell Biol 1987; 105: Mosher DF. Physiology of thrombospondin. Annu Rev Med 1990;41: Chlquet-Ehrismann R, Mackle EJ, Pearson CA, Sakakura T. Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 1986:47: Erlckson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu Rev Cell Biol 1989;5: Chlquet-Ehrismann R, Kalla P, Pearson CA, Beck K, Chlquet M. Tenascin interferes with fibronectin action. Cell 1988;53: Llghtner VA, Erlckson HP. Binding of hexabrachion (tenascin) to the extracellular matrix and its effect on cell adhesion. J Cell Sci 1990:95: Delvos U, Gajdusek C, Sage H, Harker LA, Schwartz SM. Interactions of vascular wall cells with collagen gels. Lab Invest 1982:46: Ehrlich HP, Grlswold TR, Rajaratnam JBM. Studies on vascular smooth muscle cells and dermal fibroblasts in collagen matrices: effects of heparin. Exp Cell Res 1986; 164: Lark MW, Wight TN. Modulation of proteoglycan metabolism by aortic smooth muscle cells grown on collagen gels. Arteriosclerosis 1986:6: Sakata N, Kawamura K, Takebayashl S. Effects of collagen matrix on proliferation and differentiation of vascular smooth muscle cells in vitro. Exp Mol Pathol 1990;52: Hamati HF, Brltton EL, Carey DJ. Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture. J Cell Biol 1989:108: Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis 1981;40: Campbell JH, Campbell GR. Endothelial cell influences on vascular smooth muscle phenotype. Annu Rev Physiol i 986;48: Nathan CF. Secretory products of macrophages. J Clin Invest 1987;79: Zlegler-Heltbrock HWL. The biology of the monocyte system. Eur J Cell Biol 1989;49: Samuelsson B, Goldyne M, Granstrom E, Hamberg M, Hammarstrdm S, Malmsten C. Prostaglandins and thromboxanes. Annu Rev Biochem 1978;47: McGlff JC. Prostaglandins, prostacyclin, and thromboxanes. Annu Rev Pharmacol Toxicol 1981:21: Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 1983; 220: Hammarstrdm S. Leukotrienes. Annu Rev Biochem 1983; 52: Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowlth JB. Arachidonic acid metabolism. Annu Rev Biochem 1986;55: Samuelsson B, Dahlen S-E, Lindgren ja, Rouzer CA, Serhan CN. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 1987;237: Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochem J 1989:259: Halushka PV, Mais DE, Mayeux PR, Morlnelll TA. Thromboxane, prostaglandin and leukotriene receptors. Annu Rev Pharmacol Toxicol 1989; 10: Cristol JP, Provencal B, Slrols P. Leukotriene receptors. J Recept Res 1989;9: Crooke ST, Mattern M, Sarau HM, et al. The signal transduction system of the leukotriene D 4 receptor. Trends Pharmacol Sci 1989; 10: Snyder DW, Flelsch JH. Leukotriene receptor antagonists as potential therapeutic agents. Annu Rev Pharmacol Toxicol 1989:29: Gryglewskl RJ. Prostaglandins, platelets, and atherosclerosis. Crit Rev Biochem 1980;7: Moncada S. Prostacyclin and arterial wall biology. Arteriosclerosis 1982;2:

19 984 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER Majerus PW. Arachidonate metabolism in vascular disorders. J Clin Invest 1983;72: Smith WL. Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu Rev Physiol 1986;48: Fitzgerald GA, Catella F, Oates JA. Eicosanoid biosynthesis in human cardiovascular disease. Hum Pathol 1987; 18: Lagarde M, Gualde N, Rigaud M. Metabolic interactions betwen eicosanoids in blood and vascular cells. Biochem J 1989;257: Pomerantz KB, Hajjar DP. Eicosanoids in regulation of arterial smooth muscle cell phenotype, proliferative capacity, and cholesterol metabolism. Arteriosclerosis 1989;9: Sjolund M, Nilsson J, Palmberg L, Thyberg J. Phenotype modulation in primary cultures of arterial smooth-muscle cells: dual effect of prostaglandin E,. Differentiation 1984; 27: Hajjar DP, Weksler BB, Falcone DJ, Hefton JM, Tack- Goldman K, Mlnlck CR. Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells. J Clin Invest 1982;70: Etingln OR, Weksler BB, Hajjar DP. Cholesterol metabolism is altered by hydrolytic metabolites of prostacyclin in arterial smooth muscle cells. J Lipid Res 1986;27: Palmberg L, Claesson H-E, Thyberg J. Effects of leukotrienes on phenotypic properties and growth of arterial smooth muscle cells in primary culture. J Cell Sci 1989;93: (errata vol 94, part 3) 187. Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 1984;53: Pegg AE. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 1986;234: Heby O, Luk GD, Schlndler J. Polyamine synthesis inhibitors act as both inducers and suppressors of cell differentiation. In: McCann PP, Pegg AE, Sjoerdsma A, eds. Inhibition of polyamine metabolism: biological significance and basis for new therapies. New York: Academic Press, 1987; Thyberg J, Fredholm BB. Modulation of arterial smooth muscle cells from contractile to synthetic phenotype requires induction of ornithine decarboxylase activity and polyamine synthesis. Exp Cell Res 1987,170: Thyberg J, Fredholm BB. Induction of ornithine decarboxylase activity and putrescine synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor. Exp Cell Res 1987; 170: Ewton DZ, Erwin BG, Pegg AE, Florlnl JR. The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts. J Cell Physiol 1984; 120: Joklnen MP, Clarkson TB, Prlchard RW. Animal models in atherosclerosis research. Exp Mol Pathol 1985;42: Goldberg ID, Stemerman MB, Schnipper LE, Ransll BJ, Crooks GW, Fuhro RL. Vascular smooth muscle cell kinetics: a new assay for studying patterns of cellular proliferation in vivo. Science 1979;205: Clowes AW, Reldy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49: Lamb MA, Manning JE, Reddlck RL, Grlggs TR. Smooth muscle cell proliferation in response to endothelial injury in coronary arteries of normal and von Willebrand's disease swine. Arteriosclerosis 1984;4: Ross R, Glomset J, Karlya B, Harker L. A plateletdependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci USA 1974;71: Rutherford RB, Ross R. Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate. J Cell Biol 1976;69: Ross R, Nlst C, Karlya B, Rlvest MJ, Raines E, Callls J. Physiological quiescence in plasma-derived serum: influence of platelet-derived growth factor on cell growth in culture. J Cell Physiol 1978;97: Kohler N, Llpton A. Platelets as a source of fibroblast growth-promoting activity. Exp Cell Res 1974;87: Westermark B, Wasteson A. A platelet factor stimulating human normal glial cells. Exp Cell Res 1976;98: Antonlades HN, Scher CD, Stiles CD. Purification of human platelet-derived growth factor. Proc Natl Acad Sci USA1979;76: Heldln C-H, Westermark B, Wasteson A. Platelet-derived growth factor: purification and partial characterization. Proc Natl Acad Sci USA 1979;76: Deuel TF, Huang JS, Proffitt RT, Baenziger JU, Chang D, Kennedy BB. Human platelet-derived growth factor: purification and resolution into two active protein fractions. J Biol Chem 1981;256: Raines EW, Ross R. Platelet-derived growth factor. I. High yield purification and evidence for multiple forms. J Biol Chem 1982;257: Heldin C-H, Wasteson A, Westermark B. Platelet-derived growth factor. Mol Cell Endocrinol 1985;39: Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 1986;46: Deuel TF. Polypeptide growth factors: roles in normal and abnormal cell growth. Annu Rev Cell Biol 1987;3: Hannink M, Donoghue DJ. Structure and function of platelet-derived growth factor (PDGF) and related proteins. Biochim Biophys Acta 1989;989: Heldln C-H, Westermark B. Platelet-derived growth factor: three isoforms and two receptor types. Trends Genet 1989;5: Ross R, Bowen-Pope DF, Raines EW. Platelet-derived growth factor and its role in health and disease. Philos Trans R Soc Lond [Biol] 1990;327: Doollttle RF, Hunkaplller MW, Hood LE, et al. Simian sarcoma one gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science 1983;221: Waterfield MD, Scrace GT, Whittle N, et al. Plateletderived growth factor is structurally related to the putative transforming protein p28 sls of simian sarcoma virus. Nature 1983;304: Betsholtz C, Johnsson A, Heldin C-H, et al. cdna sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature 1986;320: Heldin C-H, Backstrom G., Ostman A, et al. Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types. EMBO J 1988; 7: Hart CE, Forstrom JW, Kelly JD, et al. Two classes of PDGF receptor recognize different isoforms of PDGF. Science 1988;240: Williams LT. Signal transduction by the platelet-derived growth factor receptor. Science 1989;243: Westermark B, Claesson-Welsh L, Heldln C-H. Structural and functional aspects of the receptors for plateletderived growth factor. Progr Growth Factor Res 1989;1: Heldin C-H, Westermark B, Wasteson A. Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc Natl Acad Sci USA 1981; 78: Bowen-Pope DF, Ross R. Platelet-derived growth factor. II. Specific binding to cultured cells. J Biol Chem 1982;257: Williams LT, Tremble P, Antoniades HN. Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable. Proc Natl Acad Sci USA 1982;79: Nilsson J, Thyberg J, Heldin C-H, Westermark B, Wasteson A. Surface binding and internalization of platelet-derived growth factor in human fibroblasts. Proc Natl Acad Sci USA 1983;80:

20 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al Rosenfeld ME, Bowen-Pope DF, Ross R. Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation. J Cell Physiol 1984;121: Weinstein R, Stemerman MB, Maciag T. Hormonal requirements for growth of arterial smooth muscle cells in vitro: an endocrine approach to atherosclerosis. Science 1981;212: Nllsson J, Kslazek T, Heldln C-H, Thyberg J. Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells: differences between cells from young and adult animals. Exp Cell Res 1983:145: Clemmons DR. Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication. J Cell Physiol 1984:121: Pfeifle B, Boeder H, Dltschuneit H. Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells. Endocrinology 1987,120: Banskota NK, Taub R, Zellner K, King GL. Insulin, insulin-like growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells. Mol Endocrinol 1989;3: Clemmons DR. Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-c. Endocrinology 1985; 117: Clemmons DR, Van Wyk JJ. Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J Clin Invest 1985:75: Hosang M, Rouge M. Human vascular smooth muscle cells have at least two distinct PDGF receptors and can secrete PDGF-AA. J Cardiovasc Pharmacol 1989;14(suppl 6):S22-S Sjolund M, Rahm M, Claesson-Welsh L, Sejersen T, Heldln C-H, Thyberg J. Expression of PDGF a- and ^-receptors in rat arterial smooth muscle cells is phenotype and growth state dependent. Growth Factors (in press) 233. Grotendorst GR, Seppa HEJ, Klelnman HK, Martin GR. Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor. Proc Natl Acad Sci USA 1981:78: Grotendorst GR, Chang T, Seppa HEJ, Klelnman HK, Martin GR. Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J Cell Physiol 1982:113: Bernstein LR, Antoniades H, Zetter BR. Migration of cultured vascular cells in response to plasma and plateletderived factors. J Cell Sci 1982:56: Carpenter G, Cohen S. Epidermal growth factor. Annu Rev Biochem 1979:48: Schlessinger J, Schreiber AB, Levi A, Lax I, Libermann T, Yarden Y. Regulation of cell proliferation by epidermal growth factor. Crit Rev Biochem 1983:14: Carpenter G, Zendegui JG. Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 1986; 164: Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 1989; 58: Klagsbrun M, Edelman ER. Biological and biochemical properties of fibroblast growth factors: implications for the pathogenesis of atherosclerosis. Arteriosclerosis 1989;9: Gospodarowicz D, Hlrabayashi K, Giguere L, Tauber J-P. Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture. J Cell Biol 1981;89: Grosenbaugh DA, Amoss MS, Hood DM, Morgan SJ, Williams JD. Epidermal growth factor-mediated effects on equine vascular smooth muscle cells. Am J Physiol 1988; 255:C447-C Scott-Burden T, Reslnk TJ, Baur U, Burgin M, Biihler FR. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension 1989; 13: Winkles JA, Friesel R, Burgess WH, et al. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor). Proc Natl Acad Sci USA 1987;84: Gospodarowicz D, Ferrara N, Haaparanta T, Neufeld G. Basic fibroblast growth factor: expression in cultured bovine vascular smooth muscle cells. Eur J Cell Biol 1988;46: Le J, Vilcek J. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Invest 1987;56: Martin M, Resch K. Interleukin 1: more than a mediator between leukocytes. Trends Pharmacol Sci 1988;9: Moses HL, Coffey RJ Jr, Leof EB, Lyons RM, Keskl-Oja J. Transforming growth factor p regulation of cell proliferation. J Cell Physiol 1987;Suppl 5: Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 1987;105: Libby P, Warner SJC, Friedman GB. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest 1988:81: Bonin PD, Fici GJ, Singh JP. lnterleukin-1 promotes proliferation of vascular smooth muscle cells in coordination with PDGF or a monocyte derived growth factor. Exp Cell Res 1989; 181: Raines EW, Dower SK, Ross R. lnterleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science 1989;243: Assoian RK, Sporn MB. Type /3 transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol 1986:102: Chen J-K, Hoshi H, McKeehan WL. Transforming growth factor type /3 specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells. Proc Natl Acad Sci USA 1987:84: Majack RA. Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures. J Cell Biol 1987; 105: Goodman LV, Majack RA. Vascular smooth muscle cells express distinct transforming growth factor-/3 receptor phenotypes as a function of cell density in culture. J Biol Chem 1989;264: Owens GK, Geisterfer AAT, Wei-Hwa Yang Y, Komoriya A. Transforming growth factor-/3-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol 1988;107: Nabata T, Morimoto S, Koh E, Shiraishi T, Ogihara T. lnterleukin-6 stimulates c-myc expression and proliferation of cultured vascular smooth muscle cells. Biochem Int 1990;20: Loppnow H, Libby P. Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6. J Clin Invest 1990,85: Seifert RA, Schwartz SM, Bowen-Pope DF. Developmentally regulated production of platelet-derived growth factorlike molecules. Nature 1984;311: Nilsson J, Sjolund M, Palmberg L, Thyberg J, Heldin C-H. Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein. Proc Natl Acad Sci USA 1985:82:

21 986 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER Sejersen T, Betsholtz C, Sjolund M, Heldin C-H, Westermark B, Thyberg J. Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein. Proc Natl Acad Sci USA 1986;83: Walker LN, Bowen-Pope DF, Ross R, Reidy MA. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci USA 1986; 83: Majesky MW, Benditt EP, Schwartz SM. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/sis genes in rat aortic smooth muscle cells. Proc Natl Acad Sci USA 1988;85: McCaffrey TA, Nicholson AC, Szabo PE, Weksler ME, Weksler BB. Aging and arteriosclerosis: the increased proliferation of arterial smooth muscle cells isolated from old rats is associated with increased platelet-derived growth factor-like activity. J Exp Med 1988; 167: Sarzanl R, Brecher P, Chobanlan AV. Growth factor expression in aorta of normotensive and hypertensive rats. J Clin Invest 1989;83: Barrett TB, Benditt EP. sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci USA 1987;84: Barrett TB, Benditt EP. Platelet-derived growth factor gene expression in human atherosclerotic plaques and normal artery wall. Proc Natl Acad Sci USA 1988;85: Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mrna detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest 1988;82: Libby P, Warner SJC, Salomon RN, Birinyl LK. Production of platelet-derived growth factor-like mitogen by smooth-muscle cells from human atheroma. N Engl J Med 1988:318: Valente AJ, Delgado R, Metter JD, et al. Cultured primate aortic smooth muscle cells express both the PDGF-A and PDGF-B genes but do not secrete mitogenic activity or dimeric platelet-derived growth factor protein. J Cell Physiol 1988:136: Sjolund M, Hedin U, Sejersen T, Heldin C-H, Thyberg J. Arterial smooth muscle cells express platelet-derived growth factor (PDGF) A chain mrna, secrete a PDGF-like mitogen, and bind exogenous PDGF in a phenotype- and growth state-dependent manner. J Cell Biol 1988; 106: Naftilan AJ, Pratt RE, Dzau VJ. Induction of plateletderived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest 1989;83: Majack RA, Majesky MW, Goodman LV. Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-/?. J Cell Biol 1990; 111: Hosang M. Suramin binds to platelet-derived growth factor and inhibits its biological activity. J Cell Biochem 1985;29: Sjolund M, Thyberg J. Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle cells but does not interfere with autocrine stimulation of DNA synthesis. Cell Tissue Res 1989;256: Libby P, Ordovas JM, Birlnyi LK, Auger KR, Dinarello CA. Inducible interleukin-1 gene expression in human vascular smooth muscle cells. J Clin Invest 1986;78: Warner SJC, Auger KR, Libby P. Human interleukin 1 induces interleukin 1 gene expression in human vascular smooth muscle cells. J Exp Med 1987;165: Resink T, Hahn AWA, Scott-Burden T, Powell J, Weber E, Buhler FR. Inducible endothelin mrna expression and peptide secretion in cultured human vascular smooth muscle cells. Biochem Biophys Res Commun 1990; 168: Morisaki N, Kanzakl T, Koshikawa T, Saito Y, Yoshida S. Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor, by cultured rabbit aortic smooth muscle cells. FEBS Lett 1988;230: Morisaki N, Koyama N, Mori S, et al. Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells. Atherosclerosis 1989;78: Blaes N, Boissel J-P. Growth-stimulating effect of catecholamines on rat aortic smooth muscle cells in culture. J Cell Physiol 1983;116: Bauch H-J, Griinwald J, Vischer P, Gerlach U, Hauss WH. A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis. Exp Pathol 1987;31: Nakaki T, Nakayama M, Yamamoto S, Kato R. a,-adrenergic stimulation and /3 2 -adrenergic inhibition of DNA synthesis in vascular smooth muscle cells. Mol Pharmacol 1990;37: Majesky MW, Daemens MJAP, Schwartz SM. a,-adrenergic stimulation of platelet-derived growth factor A-chain gene expression in rat aorta. J Biol Chem 1990;265: Berk BC, Alexander RW, Brock TA, Gimbrone MA Jr, Webb RC. Vasoconstriction: a new activity for plateletderived growth factor. Science 1986;232: Berk BC, Brock TA, Webb RC, et al. Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction. J Clin Invest 1985;75: Berk BC, Alexander RW. Vasoactive effects of growth factors. Biochem Pharmacol 1989,38: Nemecek GM, Coughlin SR, Handley DA, Moskowitz MA. Stimulation of aortic smooth muscle cell mitogenesis by serotonin. Proc Natl Acad Sci USA 1986;83: Gunther S, Alexander RW, Atkinson WJ, Gimbrone MA Jr. Functional angiotensin II receptors in cultured vascular smooth muscle cells. J Cell Biol 1982;92: Paglin S, Stukenbrok H, Joyce NC, Jamleson JD. Interaction of angiotensin II with functional smooth muscle cells in culture. Am J Physiol 1987;253:C872-C Smith JB, Smith L, Brown ER, et al. Angiotensin II rapidly increases phosphatidate-phosphoinositide synthesis and phosphoinositide hydrolysis and mobilizes intracellular calcium in cultured arterial muscle cells. Proc Natl Acad Sci USA1984;81: Grlendllng KK, Rlttenhouse SE, Brock TA, et al. Sustained diacylglycerol formation from inositol phospholipids in angiotensin ll-stimulated vascular smooth muscle cells. J Biol Chem. 1986;261: Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Glnard B. Angiotensin II induces c-fos mrna in aortic smooth muscle: role of Ca 2+ mobilization and protein kinase C activation. J Biol Chem 1989,264: Gelsterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 1988;62: Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin ll-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension 1989; 13: Zachary I, Woll PJ, Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol 1987;124: Dalsgaard C-J, Hultgardh-Nilsson A, Haegerstrand A, Nilsson J. Neuropeptides as growth factors. Possible roles in human diseases. Regul Pept 1989;25:1-9

22 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al Nilsson J, von Euler AM, Dalsgaard C-J. Stimulation of connective tissue cell growth by substance P and substance K. Nature 1985;315: Payan DG. Receptor-mediated mitogenic effects of substance P on cultured smooth muscle cells. Biochem Biophys Res Commun 1985; 130: Nilsson J, Sejersen T, Hultgardh-Nilsson A, Dalsgaard C-J. DNA synthesis induced by the neuropeptide substance K correlates to the level of myc-gene transcripts. Biochem Biophys Res Commun 1986; 137: Hultgardh-Nilsson A, Nilsson J, Jonzon B, Dalsgaard C-J. Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A. J Cell Physiol 1988;137: Yanaglsawa M, Kurihara H, Klmura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332: Inoue A, Yanagisawa M, Klmura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 1989;86: Yanaglsawa M, Masaki T. Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 1989; 10: Yanagisawa M, Masaki T. Endothelin, a novel endothelium-derived peptide: pharmacological activities, regulation and possible roles in cardiovascular control. Biochem Pharmacol 1989;38: Clozel M, Flschll W, Guilty C. Specific binding of endothelin on human vascular smooth muscle cells in culture. J Clin Invest 1989;83: Takuwa Y, Kasuya Y, Takuwa N, et al. Endothelin receptor is coupled to phospholipase C via a pertussis toxininsensitive guanine nucleotide-binding regulatory protein in vascular smooth muscle cells. J Clin Invest 1990;85: Komuro I, Kurihara H, Sugiyama T, Takaku F, Yazaki Y. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988;238: Bobik A, Grooms A, Millar JA, Mitchell A, Grinpukel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol 1990;258:C408-C Araki S, Kawahara Y, Kariya K, Sunako M, Fukazaki H, Takai Y. Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells. Biochem Biophys Res Commun 1989;159: Griendllng KK, Tsuda T, Alexander RW. Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Biol Chem 1989;264: Hirata Y, Takagi Y, Fukuda Y, Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989;78: Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T. A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Biol Chem 1989;264: Brown KD, Littlewood CJ. Endothelin stimulates DNA synthesis in Swiss 3T3 cells: synergy with polypeptide growth factors. Biochem J 1989;263: Wickremaslnghe RG. The role of prostaglandins in the regulation of cell proliferation. Prostaglandins Leukot Essent Fatty Acids 1988;31: Rana RS, Hokln LE. Role of phosphoinositides in transmembrane signaling. Physiol Rev 1990;70: Huttner JJ, Gwebu ET, Panganamala RV, et al. Fatty acids and their prostaglandin derivatives: inhibitors of proliferation in aortic smooth muscle cells. Science 1977; 197: Cornwell DG, Huttner JJ, Milo GE, Panganamala RV, Sharma HM, Geer JC. Polyunsaturated fatty acids, vitamin E, and the proliferation of aortic smooth muscle cells. Lipids 1979; 14: Pietila K, Moilanen T, Nikkari T. Prostaglandins enhance the synthesis of glycosaminoglycans and inhibit the growth of rabbit aortic smooth muscle cells in culture. Artery 1980;7: Smith DL, Willis AL, Mahmud I. Eicosanoid effects on cell proliferation in vitro: relevance to atherosclerosis. Prostaglandins Leukotrienes Med 1984; 16: Loesberg C, Van Wijk R, Zandbergen J, et al. Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E,. Exp Cell Res 1985; 160: Owen NE. Effect of prostaglandin E, on DNA synthesis in vascular smooth muscle cells. Am J Physiol 1986;250: C584-C Uehara Y, Ishimitsu T, Kimura K, Ishii M, Ikeda T, Sugimoto T. Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats. Prostaglandins 1988;36: Morisaki N, Kanzaki T, Motoyama N, Saito Y, Yoshida S. Cell cycle-dependent inhibition of DNA synthesis by prostaglandin l 2 in cultured rabbit aortic smooth muscle cells. Atherosclerosis 1988;71: Morisaki N, Kanzaki T, Saito Y, Yoshida S. Lack of inhibition of DNA synthesis by prostaglandin l 2 in cultured intimal smooth muscle cells from rabbits. Atherosclerosis 1988:73: Nilsson J, Olsson AG. Prostaglandin E, inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor. Atherosclerosis 1984;53: Jonzon B, Nilsson J, Fredholm BB. Adenosine receptormediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells. J Cell Physiol 1985;124: Larrue J, Rigaud M, Da ret D, Demond J, Durand J, Bricaud H. Prostacyclin production by cultured smooth muscle cells from atherosclerotic rabbit aorta. Nature 1980; 285: Larrue J, Daret D, Demond-Henri J, Allieres C, Bricaud H. Prostacyclin synthesis by proliferative aortic smooth muscle cells: a kinetic in vivo and in vitro study. Atherosclerosis 1984;50: Kanemaru Y, Noguchi T, Kazama Y-l, Wakasugi M, Onaya T, Yoshida Y. Increased production of prostacyclin (PGI 2 ) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta. Prostaglandins 1988;36: Coughlin SR, Moskowitz MA, Zetter BR, Antoniades HN, Levine L. Platelet-dependent stimulation of prostacyclin synthesis by platelet-derived growth factor. Nature 1980; 288: Coughlin SR, Moskowitz MA, Antoniades HN, Levine L. Serotonin receptor-mediated stimulation of bovine smooth muscle cell prostacyclin synthesis and its modulation by platelet-derived growth factor. Proc Natl Acad Sci USA 1981 ;78: Shier WT. Serum stimulation of phospholipase A 2 and prostaglandin release in 3T3 cells is associated with platelet-derived growth-promoting activity. Proc Natl Acad Sci USA 1980:77: Rozengurt E, Stroobant P, Waterfield MD, Deuel TF, Keehan M. Platelet-derived growth factor elicits cyclic AMP accumulation in Swiss 3T3 cells: role of prostaglandin production. Cell 1983:34: Habenicht AJR, Goerig M, Grulich J, et al. Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase. J Clin Invest 1985,75: Habenicht AJR, Dresel HA, Goerig M, et al. Low density lipoprotein receptor-dependent prostaglandin synthesis in Swiss 3T3 cells stimulated by platelet-derived growth factor. Proc Natl Acad Sci USA 1986;83:

23 988 ARTERIOSCLEROSIS VOL 10, No 6, NOVEMBER/DECEMBER Goerig M, Habenicht AJR, Zeh W, et al. Evidence for coordinate, selective regulation of eicosanoid synthesis in platelet-derived growth factor-stimulated 3T3 fibroblasts and in HL-60 cells induced to differentiate into macrophages or neutrophils. J Biol Chem 1988;263: Lin AH, Bienkowski MJ, Gorman RR. Regulation of prostaglandin H synthase mrna levels and prostaglandin biosynthesis by platelet-derived growth factor. J Biol Chem 1989;264: Blay J, Hollenberg MD. Epidermal growth factor stimulation of prostacyclin production by cultured aortic smooth muscle cells: requirement for increased cellular calcium levels. J Cell Physiol 1989:139: Palmberg L, Claesson H-E, Thyberg J. Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells. J Cell Sci 1987;88: Baud L, Sraer J, Perez J, Nivez M-P, Ardaillou R. Leukotriene C 4 binds to human glomerular epithelial cells and promotes their proliferation in vitro. J Clin Invest 1985:76: Baud L, Perez J, Cherqui G, Cragoe EJ Jr, Ardaillou R. Leukotriene D 4 -induced proliferation of glomerular epithelial cells: PKC- and Na + -H + exchanger-mediated response. Am J Physiol 1989;257:C232-C Kragballe K, Desjarlais L, Voorhees JJ. Leukotriene B 4, C 4 and D 4 stimulate DNA synthesis in cultured human epidermal keratinocytes. Br J Dermatol 1985:113: Baud L, Perez J, Denis M, Ardaillou R. Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin. J Immunol 1987;138: Modat G, Muller A, Mary A, Gregoire C, Bonne C. Differential effects of leukotrienes B 4 and C 4 on bovine aortic endothelial cell proliferation in vitro. Prostaglandins 1987:33: Spector AA, Gordon JA, Moore SA. Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res 1988;27: Bailey JM, Bryant RW, Whiting J, Salata K. Characterization of 11-HETE and 15-HETE, together with prostacyclin, as major products of the cyclooxygenase pathway in cultured rat aorta smooth muscle cells. J Lipid Res 1983; 24: Clark MA, Littlejohn D, Conway TM, Mong S, Stelner S, Crooke ST. Leukotriene D 4 treatment of bovine aortic endothelial cells and murine smooth muscle cells in culture results in an increase in phospholipase A 2 activity. J Biol Chem 1986:261: Vicenzi E, Bianchi M, Salmona M, Donati MB, Poggl A, Ghezzi P. Platelet derived growth factor induces ornithine decarboxylase activity in NIH 3T3 cells. Biochem Biophys Res Commun 1985; 127: Rinehart CA Jr, Canellakis ES. Induction of ornithine decarboxylase activity by insulin and growth factors is mediated by amino acids. Proc Natl Acad Sci USA 1985; 82: Hovis JG, Stumpo DJ, Halsey DL, Blackshear PJ. Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts. J Biol Chem 1986;261: Widelitz RB, Matrlsian LM, Russell DH, Magun BE. Differential effects of lysosomotropic amines and polyamines on processing and biological activity of EGF. Exp Cell Res 1984;155: Mclntosh JR, Koonce MP. Mitosis. Science 1989;246: Rappaport R. Establishment of the mechanism of cytokinesis in animal cells. Int Rev Cytol 1986; 105: Brunk U, Schellens J, Westermark B. Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells. Exp Cell Res 1976:103: Schlesslnger J, Gelger B. Epidermal growth factor induces redistribution of actin and a-actinin in human epidermal carcinoma cells. Exp Cell Res 1981; 134: Mellstrom K, Hoglund A-S, Nlster M, Heldin C-H, Westermark B, Llndberg U. The effect of platelet-derived growth factor on morphology and motility of human glial cells. J Muscle Res Cell Motil 1983;4: Bockus BJ, Stiles CD. Regulation of cytoskeletal architecture by platelet-derived growth factor, insulin and epidermal growth factor. Exp Cell Res 1984:153: Herman B, Pledger WJ. Platelet-derived growth factorinduced alterations in vinculin and actin distribution in BALB/C-3T3 cells. J Cell Biol 1985:100: Mellstrom K, Heldin C-H, Westermark B. Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor. Exp Cell Res 1988; 177: Hammacher A, Mellstrom K, Heldin C-H, Westermark B. Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer. EMBO J 1989;8: Herman B, Roe MW, Harris C, Wray B, Clemmons D. Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells. Cell Motil Cytoskeleton 1987;8: Friedkin M, Rozengurt E. The role of cytoplasmic microtubules in the regulation of the activity of peptide growth factors. In: Weber G, ed. Advances in enzyme regulation, vol 19. Oxford: Pergamon Press, 1980: Otto AM. Microtubules and the regulation of DNA synthesis in fibroblastic cells. Cell Biol Int Rep 1982;6: Tucker RW. Role of microtubules and centrioles in growth regulation of mammalian cells. In: Dowben RM, Shay JW, eds. Cell and muscle motility, vol 3. New York: Plenum Press, 1983: Thyberg J. The microtubular cytoskeleton and the initiation of DNA synthesis. Exp Cell Res 1984;155: Nilsson J, Ksiazek T, Thyberg J. Effects of colchicine on DNA synthesis, endocytosis and fine structure of cultivated arterial smooth muscle cells. Exp Cell Res 1983;143: Castellot JJ Jr, Addonizlo ML, Rosenberg R, Karnovsky MJ. Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J Cell Biol 1981 ;90: Castellot JJ Jr, Favreau LV, Karnovsky MJ, Rosenberg RD. Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin: possible role of a platelet endoglycosidase. J Biol Chem 1982;257: Nilsson J, Ksiazek T, Thyberg J, Wasteson A. Cell surface components and growth regulation in cultivated arterial smooth muscle cells. J Cell Sci 1983;64: Fritze LMS, Reilly CF, Rosenberg RD. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol 1985:100: Herman IM, Castellot JJ Jr. Regulation of vascular smooth muscle cell growth by endothelial-synthesized extracellular matrices. Arteriosclerosis 1987;7: Fager G, Hansson GK, Ottosson P, Dahllof B, Bondjers G. Human arterial smooth muscle cells in culture: effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res 1988; 176: Clowes AW, Karnovsky MJ. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature 1977;265: Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin. Lab Invest 1985:52: Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration. Circ Res 1986:58: Edelman ER, Adams DH, Karnovsky MJ. Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury. Proc Natl Acad Sci USA 1990:87:

24 ARTERIAL SMOOTH MUSCLE CELLS Thyberg et al Castellot JJ Jr, Beeler DL, Rosenberg RD, Karnovsky MJ. Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. J Cell Physiol 1984;120: Wright TC Jr, Castellot JJ Jr, Petltou M, Lormeau J-C, Choay J, Karnovsky MJ. Structural determinants of heparin's growth inhibitory activity: interdependence of oligosaccharide size and charge. J Biol Chem 1989;264: Castellot JJ Jr, Wong K, Herman B, et al. Binding and internalization of heparin by vascular smooth muscle cells. J Cell Physiol 1985; 124: Rellly CF, Frltze LMS, Rosenberg RD. Heparin inhibition of smooth muscle cell proliferation: a cellular site of action. J Cell Physiol 1986; 129: Herbert J-M, Maffrand J-P. Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation. J Cell Physiol 1989; 138: Castellot JJ Jr, Cochran DL, Karnovsky MJ. Effect of heparin on vascular smooth muscle cells. I. Cell metabolism. J Cell Physiol 1985; 124: Rellly CF, Klndy MS, Brown KE, Rosenberg RD, Sonensheln GE. Heparin prevents vascular smooth muscle cell progression through the G, phase of the cell cycle. J Biol Chem 1989;264: Castellot JJ Jr, Pukac LA, Caleb BL, Wright TC Jr, Karnovsky MJ. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J Cell Biol 1989; 109: Zaragoza R, Battle-Tracy KM, Owen NE. Heparin inhibits Na + -H + exchange in vascular smooth muscle cells. Am J Physiol 1990;258:C46-C Majack RA, Bornsteln P. Heparin and related glycosaminoglycans modulate the secretory phenotype of vascular smooth muscle cells. J Cell Biol 1984;99: Majack RA, Bornsteln P. Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an M, 60,000 collagen. J Cell Biol 1985;100: Majack RA, Cook SC, Bornsteln P. Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J Cell Biol 1985; 101: Cochran DL, Castellot JJ Jr, Robinson JM, Karnovsky MJ. Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells. Biochim Biophys Acta 1988;967: Lankes W, Griesmacher A, Grunwald J, Schwartz- Alblez R, Keller R. A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. Biochem J 1988:251: Majack RA, Cook SC, Bornsteln P. Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Proc Natl Acad Sci USA 1986;83: Majack RA, Mildbrandt J, Dlxlt VM. Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet-derived growth factor. J Biol Chem 1987;262: Majack RA, Goodman LV, Dlxlt VM. Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J Cell Biol 1988; 106: Benitz WE, Lessler DS, Coulson JD, Bernfield M. Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor. J Cell Physiol 1986; 127: Reilly CF, Frltze LMS, Rosenberg RD. Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J Cell Physiol 1987; 131: Reilly CF, Fritze LMS, Rosenberg RD. Heparin-like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells. J Cell Physiol 1988; 136: Resink TJ, Scott-Burden T, Baur U, Biirgin M, Btihler FR. Decreased susceptibility of cultured smooth muscle cells from SHR rats to growth inhibition by heparin. J Cell Physiol 1989; 138: Pardee AB. G, events and regulation of cell proliferation. Science 1989; 246: Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem 1988:57: Hunter T. A thousand and one protein kinases. Cell 1987;50: Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986;233: Berridge MJ. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 1984;220: Hirasawa K, Nishizuka Y. Phosphatidylinositol turnover in receptor mechanism and signal transduction. Annu Rev Pharmacol Toxicol 1985;25: Majerus PW, Connolly TM, Deckmyn H, et al. The metabolism of phosphoinositide-derived messenger molecules. Science 1986;234: Whitman M, Cantley L. Phosphoinositide metabolism and the control of cell proliferation. Biochim Biophys Acta 1988;948: Shears SB. Metabolism of the inositol phosphates produced upon receptor activation. Biochem J 1989;260: Pouyssegur J. The growth factor-activatable Na + /H + exchange system: a genetic approach. Trends Biochem Sci 1985; 10: Moolenaar WH. Effects of growth factors on intracellular ph regulation. Annu Rev Physiol 1986;48: Soltoff SP, Cantley LC. Mitogens and ion fluxes. Annu Rev Physiol 1988;50: Grinstein S, Rotln D, Mason MJ. Na + /H + exchange and growth factor-induced cytosolic ph changes. Role in cellular proliferation. Biochim Biophys Acta 1989;988: Jones SD, Hall DJ, Rollins BJ, Stiles CD. Platelet-derived growth factor generates at least two distinct intracellular signals that modulate gene expression. Cold Spring Harb Symp Quant Biol 1988;53: Bravo R, Zerlal M, Toschi L, et al. Identification of growth-factor-inducible genes in mouse fibroblasts. Cold Spring Harb Symp Quant Biol 1988;53: Kaczmarek L. Protooncogene expression during the cell cycle. Lab Invest 1986;54: Schlessinger J. The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry 1988;27: Lau KHW, Farley JR, Baylink DJ. Phosphotyrosyl protein phosphatases. Biochem J 1989;257: Tonks NK, Charbonneau H. Protein tyrosine dephosphorylation and signal transduction. Trends Biochem Sci 1989; 14: Westermark B, Heldin C-H. Similar action of plateletderived growth factor and epidermal growth factor in the prereplicative phase of human fibroblasts suggests a common intracellular pathway. J Cell Physiol 1985; 124: Bottger BA, Sjolund M, Thyberg J. Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor. Cell Tissue Res 1988;252: Smith JB. Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc Natl Acad Sci USA 1983;80: Klarlund JK. Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell 1985;41: Wahl Ml, Olashaw NE, Nishibe S, Rhee SG, Pledger WJ, Carpenter G. Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase