Proc. Natl. Acad. Sci. USAVol. 83, pp. 7311-7315, October 1986Cell Biology

Production of platelet-derived growth factor-like molecules bycultured arterial smooth muscle cells accompaniesproliferation after arterial injury

(atherosclerosis/autocrine/differentiatioii)

L. N. WALKER*, D. F. BOWEN-POPE, R. Ross, AND M. A. REIDYtDepartment of Pathology, University of Washington, Seattle, WA 98195

Communicated by Gordon H. Sato, June 23, 1986

ABSTRACT The migration and proliferation of smoothmuscle cells (SMCs) within the intima of arteries followingmechanical injury is thought to be initiated by vessel wall injuryand release of growth factors, in particular the platelet-derivedgrowth factor (PDGF). However, the mechanism by whichSMC proliferation is regulated after platelet interaction withthe vessel wall has ceased is unknown. Here we show that SMCsderived from the intima of injured rat arteries (intimal SMCs)are phenotypically distinct from SMCs from unmanipulatedvessels (medial SMCs). Intimal SMCs secrete 5-fold greateramounts of PDGF-like activity into conditioned medium inculture, have fewer receptors for 'MI-labeled PDGF, and arenot mitogenically stimulated by exogenous purified PDGF.This study demonstrates that two SMC phenotypes can developin the adult rat artery and suggests that SMC proliferation invivo may be controlled, in part, by SMCs that producePDGF-like molecules.

Smooth muscle cells (SMCs) within the tunica media of adultarteries are in a quiescent state ofgrowth; for example, in therat these cells have a replication rate of approximately 0.02%(1, 2). This controlled growth can be disrupted by mechanicalinjury to the vessel wall, which induces SMC migration fromthe media into the intima, where subsequent proliferation ofmany of these cells occurs (1, 3-6). One suggestion has beenthat this migration and proliferation is initiated by theadhesion of platelets to the exposed subendothelium and thesubsequent release of growth factors, including the platelet-derived growth factor (PDGF) (7). Recent data in vivo suggestthat this pathway may not be totally responsible for SMCgrowth (5, 8, 9), and the rationale for this study, therefore,was to investigate what other mechanism may initiate theproliferation of vascular SMCs.

Previous studies of SMCs from injured and uninjuredarteries have shown that there are differences in their growthproperties in culture (10, 11). SMCs grow out of explantsfrom injured arteries more rapidly than from uninjuredvessels and will also grow in the presence of low concentra-tions of serum (10, 11). Another condition under whicharterial SMC proliferation is increased in vivo is in the younggrowing animal (12, 13). SMCs isolated from the aortas ofyoung rats are capable of producing PDGF-like molecules inculture (14). If intimal SMCs isolated from injured adultarteries also produce endogenous mitogens, this may explaintheir altered growth properties. We have therefore examinedintimal SMCs for production of PDGF-like activity in cultureand have compared them with SMCs from uninjured vessels(medial SMCs).

MATERIALS AND METHODSCells. The left carotid artery of 3-month-old Sprague-Daw-

ley rats was mechanically injured by passing an inflatableembolectomy catheter (size 2F) along the length of the vesselfour times (1, 6). After 2 weeks the left and right carotidarteries were removed for isolation of SMCs. The rightcarotid artery (Fig. lA) was stripped of adventitia andendothelium, and medial SMCs were obtained by digestionwith elastase and collagenase for 2 hr. To obtain intimalSMCs, the intimal thickening (Fig. 1B) was stripped from theleft carotid artery (Fig. 1C), by using fine forceps undermagnification, and was then digested with collagenase andelastase. Both intimal and medial SMCs were routinelygrown in Waymouth's MB 752/1 medium supplemented with10% fetal bovine serum.Morphology. The morphology of confluent cultures of

medial and intimal SMCs was examined by phase-contrastlight microscopy and scanning electron microscopy. Cells forscanning electron microscopy were grown to confluence onglass coverslips, washed with phosphate-buffered saline, andfixed overnight with 2% glutaraldehyde/1% paraformalde-hyde/0.1 M phosphate buffer, pH 7.4. Coverslips were thendehydrated in increasing concentrations of ethanol and crit-ical-point-dried using liquid carbon dioxide. Dried coverslipswere mounted on aluminum stubs with conductive paste,sputter-coated with gold/palladium, and viewed at 15 kV ina JEOL JSM 35 scanning electron microscope.

Cell Identification. von Willebrand factor. Cells weregrown to confluence on glass slides, washed with phosphate-buffered saline, and fixed for 30 min with cold methanolcontaining 0.3% hydrogen peroxide. Slides were then incu-bated with rabbit anti-human von Willebrand factor for 30min, followed by biotinylated goat anti-rabbit IgG for 30 min.The biotinylated probe was then reacted with avidin-biotincomplex and 3,3'-diaminobenzidine. Rat endothelium wasused as a positive control for von Willebrand factor staining;nonimmune rabbit serum was used as a negative control.Acetylated low density lipoprotein. Confluent cultures

were incubated with acetylated low density lipoprotein(AcLDL) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetrameth-ylindocarbocyanine perchlorate (dil-AcLDL, 10 ,ug/ml), for4 hr at either 40C or 370C. Cells were rinsed with freshmedium and were observed by fluorescence microscopy(excitation 514 nm, emission 550 nm). Bovine endotheliumwas used as a positive control for the presence of receptorsfor AcLDL.

Abbreviations: PDGF, platelet-derived growth factor; SMC, smoothmuscle cell; dil-AcLDL, acetylated low density lipoprotein labeledwith 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per-chlorate.*Present address: Lions Eye Institute, Queen Elizabeth II MedicalCentre, Nedlands, West Australia 6009.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. In vivo source of cells for isolation. (A) Medial SMCs (m)were obtained from the right carotid artery. (B) Intimal SMCs (i) wereobtained from the left carotid artery 2 weeks after mechanical injuryto induce intimal proliferation. (C) A partially dissected carotidartery with some residual intimal smooth muscle cells (i) left attachedto the vessel wall.

Smooth muscle actin. Cells were grown to confluence onglass slides and SMC-specific actin was identified using amonoclonal antibody against SMC a-actin (15). Immunocyto-chemical staining was as described above, except that thesecondary antibody was biotinylated horse anti-mouse IgG,and the negative control was nonimmune horse serum.

Production of PDGF-Like Activity. Primary SMC culturesfrom individual animals were passaged from 2-cm2 wells into25-cm2 flasks containing Dulbecco's modified Eagle's medi-um (DMEM) supplemented with 20% fetal bovine serum.When cells were almost confluent, the medium was replaced

with DMEM supplemented with 2% fetal bovine plasma-derived serum, which was discarded and replaced with freshmedium after 24 hr. After 48 hr the conditioned medium wascollected and assayed for PDGF-like activity by competitivebinding in a radioreceptor assay, as described (16). Cellnumber was determined at the end of the 48-hr conditioningperiod by trypsinization and electronic particle counting.PDGF-like activity in conditioned medium was also deter-mined after incubation with various concentrations of goatanti-human PDGF IgG or nonimmune goat IgG for 2 hr at 40C.Residual PDGF-like activity in the conditioned medium wasthen determined as described above.PDGF Receptors. After two passages, cells were trypsin-

ized and plated in 24-well trays at 2 x 104 cells per well inDMEM supplemented with 20% fetal bovine serum. Whencells were almost confluent, the medium was replaced withDMEM supplemented with 2% fetal bovine plasma-derivedserum. Forty-eight hours later, cells from triplicate wellswere assayed for specific binding of 1251-labeled PDGF asdescribed (16).Response to PDGF. Second-passage medial and intimal

SMCs were trypsinized and plated in 24-well plates at 1.5 x104 and 3 x 104 cells per well, respectively (to allow fordifferences in plating efficiency), in Waymouth's mediumsupplemented with 2% fetal bovine plasma-derived serum.After 24 hr the medium was replaced, with Waymouth'smedium supplemented with either 2% fetal bovine plasma-derived serum, 2% plasma-derived serum plus 10 ng ofpurified human PDGF per ml, or 10% fetal bovine serum. Cellcounts were determined from triplicate wells at various timeintervals, using electronic cell counting.

Materials. Goat anti-human von Willebrand factor waspurchased from Dako (Santa Barbara, CA). Biotinylatedantibodies and avidin-biotin complex were purchased fromVector Laboratories (Burlingame, CA). DiI-AcLDL waspurchased from Biomedical Technologies (Cambridge, MA).Monoclonal antibodies to smooth muscle a-actin were pro-vided by A. Gown (Department of Pathology, University ofWashington). Purified human PDGF (17) and 251I-labeledPDGF (16) were prepared according to published procedures.Fetal bovine plasma-derived serum was purchased fromHyclone Laboratories (Logan, UT) and did not containPDGF as assayed by competitive binding in the radioreceptorassay. A preparation of goat anti-human PDGF IgG was usedto abolish PDGF-induced mitogenesis and PDGF binding toresponsive cells (14, 18).

RESULTSMorphology and Identification. In this study we have

isolated two phenotypes of arterial SMC, medial and intimal,from quiescent (Fig. lA) and proliferating (Fig. 1B) popula-tions of SMCs, respectively. SMC proliferation was inducedby injury to the rat carotid artery with a balloon catheter,which induces an approximate doubling of cell number in 2weeks (Fig. 1B). These intimal cells could be readily sepa-rated from the medial cells by simple dissection (Fig. 1C).Medial and intimal SMCs exhibited two distinct morpholog-ical phenotypes in culture. Medial SMCs were spindle-shaped cells which showed extensive overlapping and formedhills and valleys when confluent (Fig. 2A). In contrast,intimal SMCs grew mainly as a monolayer with minimaloverlapping of their cell borders (Fig. 2B). Intimal and medialcells in culture were classified as SMCs, as both cell typescontained SMC-specific actin as determined by immunocyto-chemical staining with a monoclonal antibody.The morphology of intimal SMCs in culture was, however,

similar to that of endothelial cells, and the cells weretherefore tested for the endothelial cell markers von Wil-lebrand factor and AcLDL receptors. Immunocytochemical

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Table 1. Rate of production of PDGF-like activity by medial andintimal SMCs

PDGF-like activity,ng per 106 cells

Medial Intimal Ratio,Culture no. SMCs SMCs intimal/medial

1 0.63 1.01 1.602 0.11 0.46 4.183 0.09 0.56 6.204 0.06 0.48 8.005 0.20 0.63 3.156 0.04 0.59 14.807 0.44 0.90 2.058 0.23 0.41 1.789 0.08 0.43 5.3810 - 1.3311 0.38 0.25 O.6612 0.06 0.34 5.70Mean ± SD 0.21 ± 0.19 0.62 ± 0.31* 4.86 ± 4.00

Conditioned (48 hr) medium collected from primary-passageintimal and medial SMCs was assayed for PDGF-like activity bycompetitive binding in a radioreceptor assay. In all cases, exceptculture no. 12, which was a pool of tissue from five animals, cultureswere derived from individual animals.*P= 0.0014.

although less so than 10o fetal bovine serum (Fig. SA). Underidentical conditions, purified PDGF was not mitogenic forintimal SMCs (Fig. 5B). The growth rate of medial and intimalSMCs in 2% plasma-derived serum was similar: intimalSMCs did not grow more rapidly than medial SMCs. IntimalSMCs were also capable of more rapid proliferation as seenby their growth in 10% fetal bovine serum.

FIG. 2. SMC morphology in vitro. The morphology of confluentcultures of medial (A) and intimal (B) SMCs was examined byphase-contrast light microscopy. (x250.)

staining, using an antibody that detected the antigen in ratendothelial cells, showed that neither intimal nor medialSMCs contained von Willebrand factor antigen. Neitherintimal nor medial SMCs expressed detectable receptors forAcLDL (dil-AcLDL was used as probe), whereas endothelialcells showed uptake both at 370C and at 40C.

Production of PDGF-Like Activity. Twelve paired intimaland medial SMC cultures were examined for production ofPDGF-like activity by competitive binding (Table 1). Allexcept one exhibited higher production ofPDGF-like activityby intimal SMCs than by medial SMCs from the same rat(Table 1). Although rates ofproduction ofPDGF-like activityvaried among individual animals, intimal cells produced anaverage of approximately 5 times more PDGF-like activitythan medial cells. This activity was found to be antigenicallyrelated to human PDGF. The PDGF-like activity in condi-tioned medium from intimal SMCs could be neutralized by anantibody against purified human PDGF (75% neutralizationby 256 ,g/ml in Fig. 3 and 95% neutralization by 200 gg/mlin a second experiment with another preparation of condi-tioned medium).PDGF Receptors and Response to PDGF. As well as

producing more PDGF-like activity than medial SMCs,intimal SMCs also showed less specific binding of 1251I-labeledPDGF than did medial SMCs (Fig. 4). Decreased numbers ofreceptors for PDGF could also be reflected by a decreasedresponse of the cells to exogenous growth factor. We there-fore compared the growth of intimal and medial SMCs in thepresence of exogenous purified PDGF (Fig. 5). In mediumsupplemented with 2% fetal bovine plasma-derived serum,purified human PDGF was mitogenic for rat medial SMCs,

DISCUSSIONOur results show that two phenotypes of arterial SMCs,medial (quiescent) and intimal (proliferating), may exist in theadult rat vessel, the intimal phenotype being induced orselected as a response to mechanical injury. Intimal andmedial SMCs exhibited different morphologies in culture.Intimal SMC morphology in culture closely resembled that

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FIG. 3. Neutralization of PDGF-like activity in conditionedmedium by antibodies against human PDGF. Conditioned mediumfrom cultures of intimal SMC was incubated with various concen-trations of goat anti-human PDGF IgG (o) or nonimmune goat IgG(e). Residual PDGF-like activity in the conditioned medium was thendetermined by radioreceptor assay and was expressed as a percent-age of the activity determined in the absence of IgG.

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'251 labeled PDGF, ng/mI

FIG. 4. Specific binding of 1251-labeled PDGF to medial andintimal SMCs. Confluent cells, which had been maintained inmedium containing 2%o fetal bovine plasma-derived serum for 72 hr,were assayed for specific binding of 1251-labeled PDGF. Valuesshown are averages from triplicate wells of medial SMCs isolatedfrom three individual animals (closed symbols) and intimal SMCsisolated from six individual animals (open symbols).

seen in vivo in injured arteries when luminal SMCs line thevessel instead of endothelial cells (4, 5). No differences inmorphology have previously been reported for SMCs cul-tured from mechanically injured arteries (10, 11) or fromarteries where intimal proliferation has been induced bydietary manipulation (19, 20). In both cases, however, cellswere derived from explants of the complete vessel thicknessrather than the intima alone, so providing a mixed populationof starting cells.

In the present study, the morphology of intimal SMCs issimilar to that of arterial endothelial cells. It is unlikely thatthe cultures of intimal SMCs in these studies were contain-

inated by endothelial cells, as the segment of artery fromwhich cells were derived lacks endothelium (4). IntimalSMCs could also be distinguished from endothelial cells inthat they lacked von Willebrand factor (21), did not haveAcLDL receptors (22), expressed detectable numbers ofreceptors for PDGF (23), and contained SMC-specific actin(15).

Intimal SMCs also differed from medial SMCs in that theyproduced greater amounts of PDGF-like activity in culture.Medial SMCs from the carotid arteries appeared to producehigher basal levels of PDGF-like activity than that previouslyreported for adult rat aortic SMCs (14), suggesting that ratesof production of PDGF-like activity may be dependent on thesite in the arterial tree from which the cells are derived or onthe number of passages in culture (24).

Intimal SMCs produced, on average, 5 times as muchPDGF-like activity as medial SMCs, and this activity couldbe completely neutralized by antibodies against humanPDGF, showing that the molecules are antigenically similar.Intimal SMCs also showed less specific binding of human1251I-labeled PDGF than did medial SMCs. Growth factorproduction and decreased numbers of specific receptors havealso been described for transformed cells (18, 25, 26), and ithas been suggested that an autocrine (27, 28) system ofgrowth control may be operating whereby the secretedgrowth factor occupies and causes down-regulation of spe-cific receptors on the transformed cell surface. If this hy-pothesis is correct, then cells producing growth factorsshould be less dependent on exogenous sources of purifiedgrowth factors. To investigate this, we have examined thegrowth of intimal and medial SMCs in the presence orabsence of purified PDGF. We found that intimal SMCs werenot mitogenically stimulated by PDGF, whereas medialSMCs were. This is consistent with the hypothesis thatreduced numbers of detectable receptors for PDGF onintimal SMCs is due to receptor occupation and down-regulation by exogenously produced PDGF-like activity.However, the growth of intimal SMCs in the absence ofadded PDGF was no more rapid than that of medial SMCs,and they were not proliferating at their maximal rate. A

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FIG. 5. Response of SMCs to exogenous purified PDGF. Medial (A) and intimal (B) SMCs were plated in 24-well plates in mediumsupplemented with 2% fetal bovine plasma-derived serum. After 24 hr, the medium was replaced with fresh medium supplemented with either2% plasma-derived serum (o), 2% plasma-derived serum plus 10 ng of purified human PDGF per ml (o), or 10% fetal bovine serum (n). Cellnumber was determined at various time intervals by electronic cell counting. Similar results were obtained in five other experiments usingdifferent cultures of both intimal and medial cells.

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possible explanation is that intimal SMCs are also dependenton other factors, such as somatomedin C (29), to show amaximal response to PDGF.The observations from this study, in conjunction with

those reported in young animals (14), suggest that SMCproliferation in vivo correlates with secretion of PDGF-likemolecules in culture. Thus, bursts of SMC proliferation invivo during development in young animals and followinginjury in adults may be regulated, in part by mechanismssimilar to those seen in transformed cells.

We thank Delnora Williams for technical assistance and GinnyWejak for preparation of this manuscript. This work was funded inpart by National Institutes of Health Grants HL18645, HL30203,HL03174, and GM35501 and by a grant from R. J. ReynoldsIndustries Inc. M.A.R. is an Established Investigator from theAmerican Heart Association and is supported in part from fundscontributed by the Washington affiliate.

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