Sachin Patel, Yi Shi, Rodica Niculescu, Eugene H. Chung, Jack L. Martin and Andrew ZalewskiCharacteristics of Coronary Smooth Muscle Cells and Adventitial Fibroblasts

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Characteristics of Coronary Smooth Muscle Cells andAdventitial Fibroblasts

Sachin Patel, MD; Yi Shi, MD, PhD; Rodica Niculescu, DVM; Eugene H. Chung;Jack L. Martin, MD; Andrew Zalewski, MD.

Background—Recent findings suggesting the involvement of adventitial cells in coronary repair have raised questionsregarding the phenotypic “plasticity” of medial smooth muscle cells (SMCs). Accordingly, the aims of the present studywere to examine the characteristics of coronary medial and adventitial cells and to compare the responses of coronaryand noncoronary SMCs to stimulation.

Methods and Results—Enzymatically isolated coronary SMCs (human and porcine) were distinct from noncoronarySMCs, showing poor adhesion and spreading, as well as lower proliferation, collagen synthesis, and LDL degradation.Several extracellular matrix components (Matrigel, collagen I and IV, laminin, vitronectin, fibronectin) or growthfactors (epidermal growth factor, platelet-derived growth factor-BB, insulin growth factor-1, interleukin-1a) failed toaugment the adhesion or proliferation of coronary SMCs to the levels observed in noncoronary SMCs. Unlike coronarySMCs, coronary fibroblasts demonstrated high adhesion, proliferation, collagen synthesis, and avid LDL metabolism.Limited responses of coronary SMCs were associated with sustained expression of differentiation markers (a-smoothmuscle actin, h-caldesmon, and smooth muscle myosin heavy chain), whereas noncoronary SMCs showed markedphenotypic heterogeneity.

Conclusions—Coronary SMCs appeared to maintain highly differentiated phenotype in response to stimulation, whereascoronary adventitial fibroblasts demonstrated several characteristics that are essential during vascular repair. CoronarySMCs, however, were distinct from noncoronary medial cells, which displayed greater phenotypic heterogeneity andversatility in culture. We postulate that the mechanism of vascular repair may differ among vascular beds, pointing tothe importance of coronary artery–specific investigations in vascular biology.(Circulation. 2000;101:524-532.)

Key Words: remodelingn arteriesn muscle, smoothn cells

Several mechanical, hemodynamic, or infectious factorscontribute to the activation of resident vascular cells,

resulting in the formation of intimal lesions.1,2 This processleads to changes in the cellular composition of the vesselwall, which accompany a broad spectrum of cardiovasculardisorders.3–5 Although intimal cells share some similaritieswith medial smooth muscle (SM) cells (SMCs), they alsodisplay several distinct characteristics regarding morphology,gene expression, and synthetic properties (for a review, seeSchwartz et al5). Several concepts have emerged regardingthe mechanisms underlying the expansion of vascular cells invarious disease states.6–8 The first paradigm is based on theassumption that the cells involved in vascular repair areprimarily of SM origin. The most widely accepted viewimplies the “plasticity” of virtually all medial SMCs, whichmodulate their phenotype in response to stimulation. This hasbeen based on the observed changes in cultured SMCs rapidlylosing their contractile features and becoming “synthetic”cells.6 A second paradigm, which is based on more recent

studies, however, suggests heterogeneity of the arterial me-dia, with only a fraction of SMCs capable of a rapid responseto stimulation. These “stem” SMCs, isolated from the rataorta or carotid artery, differ in regard to morphology, geneexpression, and behavior in culture compared with the re-maining medial cells.9,10 The third paradigm implies thathighly reactive “nonmuscle” cells, being able to proliferate,migrate, and synthesize extracellular matrix proteins, are alsoinvolved in the repair of the vessel wall. Although they aresparsely distributed within vascular media (eg, carotid artery,pulmonary artery, and saphenous vein), nonmuscle fibro-blasts are abundant in the adventitia.11–13 After severe coro-nary injury (porcine model), nonmuscle cells of adventitialorigin proliferate and migrate to the luminal region duringneointimal formation.14,15 They also acquirea-SM actin(myofibroblast formation) and are capable of the synthesis ofseveral extracellular matrix proteins.16–18

Although these concepts remain the subject of ongoingdebate, they are not mutually exclusive, because regional

Received May 14, 1999; revision received July 30, 1999; accepted August 13, 1999.From the Cardiovascular Research Center (S.P., Y.S., R.N., E.H.C., A.Z.), Department of Medicine (Cardiology), Thomas Jefferson University,

Philadelphia, Pa; and Bryn Mawr Hospital (J.L.M.), Bryn Mawr, Pa.Correspondence to Andrew Zalewski, MD, or Yi Shi, MD, PhD, Thomas Jefferson University, Division of Cardiology, Suite 410N, 1025 Walnut St,

Philadelphia, PA 19107. E-mail andrew.zalewski@tju.edu or yi.shi@tju.edu© 2000 American Heart Association, Inc.

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differences in the mechanisms of arterial repair may arisefrom the diverse lineages of vascular cells.19 In particular, thecoronary vasculature demonstrates unique development,which differs from that of the aorta and its major tributar-ies.20–22The results of the present study suggest that coronarySMCs are less responsive to stimulation and exhibit lowersynthetic capability than other vascular SMCs. When thecellular constituents of the coronary arteries were analyzed,adventitial cells demonstrated particularly dynamic pheno-typic characteristics. These findings suggest there are func-tional differences in the cellular constituents of coronary andnoncoronary vascular beds, which may influence the mech-anisms of vascular repair and lesion formation.

MethodsCell Isolation and CulturePorcine arteries (epicardial coronary arteries, iliac artery, thoracicaorta) were harvested and opened, and their endothelial layers wereremoved. Human epicardial coronary arteries and samples of tho-racic aorta, which were obtained from the recipients of hearttransplantation, were prepared in an identical manner. For coronaryarteries, the adventitia and media were separated with the assistanceof surgical loops (magnification 23). The separation of adventitialand medial layers was confirmed through immunostaining witha-SM actin antibody of random samples (not shown). For othervascular beds, the adventitial layer was stripped off, and only themedia was used for cell isolation. The tissues were minced into smallpieces and repeatedly incubated with collagenase type II (1 mg/mL;Worthington) and elastase (0.5 mg/mL; Sigma) for 30 minutes at37°C with rocking. The cells were collected after passage of thedigestion solution through a filter (pore size 70mm; BectonDickinson). After the addition of 10% heat-inactivated FBS andcentrifugation, the cells were suspended in DMEM supplementedwith 10% FBS, 100 IU/mL penicillin, 100mg/mL streptomycin, and2 mmol/L glutamine. The cells from all digestions were pooled, andthe viability was estimated in random samples through trypan blueexclusion or MTT assay. Primary cells and early passage cells(between 2 and 7) were used.

Adhesion and Growth AssaysFreshly isolated cells were plated at 20 000 cells/well in 10% FBSwith or without coatings (24-well plates). After 24 hours, the wellswere rinsed, and attached cells were trypsinized and counted in aCoulter counter. The rate of adhesion was calculated as a percentageof the initially plated cells. The following coatings were used toenhance coronary SMC adhesion: Matrigel (10mg/well; BectonDickinson), fibronectin (100 ng/well; Sigma), type I collagen (22mg/well; Becton Dickinson), type IV collagen (22mg/well; BectonDickinson), laminin (10mg/well; Sigma), and vitronectin (1mg/well;Sigma). Briefly, the wells (24-well plates) were coated with 200mLof the solution at 4°C overnight with gentle rocking. The excess ofthe coating solution was then removed, and the plates were air driedfor 10 minutes. In addition, the wells that were coated with collagen,which was dissolved in 0.01 mol/L HCl, were rinsed twice withDMEM. Each experiment was carried out in triplicate and repeated3 times on different occasions with cells isolated from multipledonors. Data represent mean6SD of 6 to 9 values from 3 separateexperiments.

For growth assay, the cells were plated at 20 000 or 100 000cells/well (24-well plates) in DMEM supplemented with 10% FBS.At 2 days, cells were washed and fresh 10% FBS was added. Cellswere then trypsinized at 2, 4, 7, 9, and 14 days after plating andcounted in a Coulter counter. Because coronary SMCs did not adhereand grow under these conditions, they were plated onto Matrigel-coated surfaces, and various growth factors or their combinationswere tested in addition to 10% FBS: epidermal growth factor (EGF;75 mg/mL; kindly provided by Dr James San Antonio, Thomas

Jefferson University), platelet-derived growth factor (PDGF-BB;100 ng/mL, Sigma), insulin growth factor-1(IGF-1; 10 ng/mL;Sigma), and interleukin (IL)-1a (1 ng/mL; Sigma). Each growthexperiment was carried out in triplicate for each time point andrepeated 3 times on different occasions with cells isolated frommultiple donors. Data represent mean6SD of 9 values from 3experiments.

Protein SynthesisFor overall protein synthesis, cells were labeled with35S-methionine(50 mCi/mL; DuPont-New England Nuclear) for 40 hours (primarycells) or 24 hours (early passages). The conditioned media and 2washes were collected and subjected to TCA (10%) precipitation.After being washed twice with 5% TCA, radioactivity were deter-mined with a beta scintillation counter (Wallac). Protein synthesiswas expressed as dpm per cell number. Data represent mean6SD of6 values derived from 3 experiments.

For collagen synthesis, subconfluent primary cells (200 000 cells)and confluent cells (passages 2 to 6) were labeled with14C-proline(10 mCi/mL; DuPont-New England Nuclear) in DMEM containing10% FBS and ascorbic acid (25mg/mL) for 40 hours (primary cells)or 24 hours (confluent cells), respectively. The conditioned mediaand 2 washes were precipitated with 10% TCA at 4°C overnight.After centrifugation, the pellets were washed twice with 5% TCAand once with absolute ethanol. The pellets were dried and thenhydrolyzed overnight with 0.45 mL of 0.2 mol/L NaOH. Radiola-beled collagen was quantified according to the collagenase digestionmethod.23 Briefly, hydrolyzed samples were divided into 2 aliquots(0.2 mL each) and neutralized by the addition of 0.2 mL of 0.08 NHCl. After the addition ofN-ethylmaleimide (1.25mmol/L), CaCl2(0.25 mmol/L), and bacterial collagenases (25mg/mL Clostridiumhistolyticumtype III; Calbiochem), samples with a final volume of0.5 mL were then incubated at 37°C for 5 hours. The control sampleswere treated without the addition of collagenases. The reaction wasstopped by the addition of 0.5 mL of 20% TCA containing 0.5%tannic acid and BSA (100mg/mL). Newly synthesized collagen wascalculated as the difference between the radioactivity values of thesamples treated, with or without collagenases (dpm/cell). The datarepresent mean6SD of 6 to 9 values derived from 3 experiments.

LDL Isolation, Modification, and LabelingLDL (density, 1.020 to 1.063 g/mL) was isolated from fresh humanplasma through sequential ultracentrifugation.24,25 LDL oxidation(oxLDL) was achieved through the incubation of LDL (200mg/mL)with 5 mmol/L CuSO4 at 37°C for 2 to 3 hours. Oxidation wasmeasured by monitoring the change of the diene absorption (234 nm)and was stopped by the addition of 100mmol/L EDTA and40 mmol/L butylated hydroxytoluene. LDL was iodinated withNa-125I (2 mCi; DuPont-New England Nuclear) and eluted throughPD-10 columns with 0.15 mol/L NaCl and 1 mmol/L EDTA.26

125I-LDL was dialyzed for 48 hours at 4°C against 0.15 mol/L NaCland 1 mmol/L EDTA. The activity of125I-LDL ranged from 200 to400 cpm/ng LDL protein with.98% of the 125I radioactivityprecipitable by TCA and,5% of the125I radioactivity extractablewith chloroform-methanol.125I-LDL was sterilized by passagethrough a filter (pore size 0.45mm), stored at 4°C under argon, andused within 4 weeks.

For assessment of LDL degradation, freshly isolated cells wereplated at 100 000 cells/well in DMEM supplemented with 10%human lipoprotein–deficient serum (for native LDL) or 10% FBS(for oxLDL) for 48 hours. Then,125I-labeled native LDL or oxLDLwas added to the cells for an additional 16 hours. The conditionedmedia were collected and subjected to TCA (10%) precipitation andchloroform extraction. The radioactivity values (125I-tyramine) in theaqueous phase were counted in a gamma counter (Wallac). The cellswere lysed (0.1 mol/L NaOH), and cellular protein content wasmeasured. Nonspecific LDL degradation (in the presence of 50-foldexcess of unlabeled LDL and in cell-free wells) has been subtractedfrom all values. Data represent mean6SD of triplicate measure-ments. The experiments were repeated 3 times on separate occasionsin both primary cells and cells at passages 2 to 7.

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ImmunostainingVascular cells were plated onto coverslips with or without Matrigelcoating. Coronary SMCs were grown on coverslips coated with andwithout Matrigel. At different time points, the coverslips were fixedwith HistoChoice for 30 minutes and air dried. The coverslips werestained with Vectastain Elite ABC system (Vector Laboratories).They were incubated with primary antibodies for 1 hour at roomtemperature, followed by biotinylated secondary horse anti-mouseantibodies (1:2000; Vector Laboratories). Table 1 lists specificantibodies, the concentrations used, and their sources. Negativecontrols included either the omission of primary antibody or itsreplacement with irrelevant mouse IgG (125 ng/mL). The immuno-stains were visualized with the use of a diaminobenzidine tetrahy-drochloride substrate kit (Vector Laboratories) followed byhematoxylin counterstaining.

Statistical AnalysisData are expressed as mean6SD values. One-way ANOVA wasused to compare the multigroup variables. If theF test results weresignificant, Bonferroni’s analysis was carried out to determine thedifferences among subgroups. A value ofP,0.05 was required toreject the null hypothesis.

ResultsCell Morphology, Adhesion, and GrowthEnzymatic digestion yielded.80% viable cells regardless ofvascular bed (trypan blue exclusion and MTT assay). Asexpected, medial cells isolated from the aorta and iliac arterydisplayed a spindle-shaped morphology, followed by a typi-cal “hill-and-valley” pattern at confluence. In contrast, coro-nary SMCs demonstrated distinct morphology in culture(Figure 1). Coronary adventitial cells isolated from the samevessels, however, were spindle shaped after plating andshowed a hill-and-valley configuration at confluence, thusdisplaying morphology similar to that of noncoronary SMCs(Figure 1).

Porcine SMCs derived from noncoronary vascular mediashowed rapid adhesion to plastic, which was comparable tothat of coronary adventitial fibroblasts (Figure 2A). This

TABLE 1. Antibodies Used in the Study

Specificity Clone Concentration Supplier

a-SM actin 1A4 1;50 Sigma

h-Caldesmon hCD 1;200 Sigma

SM MHC hSM-V 1;800 Sigma

SM1 MHC 3F8 1;200 Seikagaku

SM2 MHC 1G12 1;800 Seikagaku

Figure 1. Photomicrograph showingmorphology of vascular cells derivedfrom porcine coronary and noncoronaryarteries. Coronary SMCs exhibited a rod-shaped morphology at 2 days, whichwas maintained for $10 days in culture.In contrast, coronary adventitial cells andnoncoronary medial cells (aorta and iliacartery) displayed a spindle-shaped mor-phology. They reached confluence at 7to 10 days, when a typical hill-and-valleypattern was observed (magnification1003). FIB indicates fibroblasts.

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contrasted with coronary medial SMCs, which showed sig-nificantly lower adhesion (P,0.01). Because cell attachmentis dependent on the surrounding extracellular matrix proteins,we attempted to increase coronary SM adhesion through thecoating of culture surfaces with several matrix proteins(Matrigel, collagen types I and IV, laminin, vitronectin,fibronectin). As shown in Figure 2B, different extracellularmatrix proteins, the use of EGF in the culture medium, andthe prolongation of adhesion time to 48 hours produced onlyminimal improvement in the adhesion of coronary SMCs.

As expected, porcine noncoronary SMCs exhibited rapidgrowth in the presence of 10% FBS, with exponential growthbeginning at 2 days (Figure 3A). These cells continued toreplicate after being subcultured for.10 passages andexhibited similar growth on plastic or Matrigel-coated sur-faces. In contrast, the growth response of porcine coronarySMCs was markedly slower, although coronary adventitialfibroblasts isolated from the same vessels displayed a dy-namic growth in culture (Figure 3A). These differences werenot species dependent, inasmuch as human coronary SMCsalso demonstrated lower growth capabilities (Figure 3B). Todetermine whether specific growth factors are required for

coronary SMC growth, several growth factors (EGF, PDGF-BB, IGF-1, or IL-1a) were added to culture medium contain-ing 10% FBS. Notwithstanding this stimulation, coronarySMCs continued to demonstrate limited replication in culture(Figure 3C). To exclude the possibility that their slower

Figure 2. Adhesion of vascular cells derived from coronaryarteries and noncoronary vascular beds. A, Freshly isolated por-cine cells were plated at 20 000 cells/well in 10% FBS withoutcoating, and number of attached cells was counted 24 hourslater. Cells isolated from coronary media (CA-SMC) exhibitedlower adhesion compared with cells derived from aortic media(AO-SMC), iliac artery media (IA-SMC), or coronary adventitia(CA-FIB) (*P,0.01 vs all other groups by ANOVA with Bonferro-ni’s correction). B, Coronary SMCs were plated at 20 000 cells/well (10% FBS with addition of 75 mg/mL EGF) onto Matrigel(MG), collagen type I (COL I), collagen type IV (COL IV), laminin(LAM), fibronectin (FN), or vitronectin (VN). Number of attachedcells was counted 48 hours later. Despite these modifications,coronary medial cells showed only modest improvement in celladhesion. Each column represents mean6SD (n56 to 9).

Figure 3. Growth of vascular cells derived from coronary andnoncoronary arteries. A, Enzymatically isolated porcine cellsfrom aortic media (AO-SMC), iliac artery media (IA-SMC), andcoronary adventitia (CA-FIB) and media (CA-SMC) were platedat 20 000 cells/well in 10% FBS. All cells demonstrated similargrowth rates except for coronary SMCs. B, Human aortic andcoronary cells were grown in 10% FBS (20 000 cells/well).Human coronary SMCs displayed significantly slower prolifera-tion (hCA-SMC) compared with aortic SMCs (hAO-SMC) andcoronary adventitial fibroblasts (hCA-FIB). C, Coronary SMCswere plated onto Matrigel-coated surface and grown in 10%FBS with addition of 75 mg/mL EGF, 100 ng/mL PDGF-BB, 10ng/mL IGF-1, or 1 ng/mL IL-1a. Despite different stimuli, coro-nary medial cells exhibited slow growth rates. Each point repre-sents mean6SD (n59).

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growth was due to low density, porcine coronary SMCs werealso plated at high density (100 000 cells/well), which failedto increase coronary SMC growth or their ability to reachconfluence after.30 days in culture (not shown).

Metabolic Labeling, Collagen Synthesis, andLDL MetabolismTo compare metabolic activity in different vascular cells,overall protein synthesis was assessed by35S-methioninelabeling. No major differences were observed in total proteinsynthesis, which was consistent with similar viability ofprimary porcine cultures (Figure 4A,P5NS). Because “syn-thetic” phenotype of SMCs is marked by an increase inextracellular matrix synthesis, de novo collagen synthesis wasmeasured with the use of14C-proline incorporation, followedby collagenase digestion. Noncoronary SMCs from the aortaand iliac artery showed comparable collagen synthesis,whereas coronary medial SMCs produced significantly lesscollagen (Figure 4B,P,0.001). Coronary adventitial fibro-blasts from the same coronary arteries also exhibited a higherability to synthesize collagen than coronary SMCs(P,0.001).

The differences among vascular cells raised the questionregarding regional LDL metabolism. To this end, the ability

of vascular cells to degrade LDL was determined. CoronarySMCs demonstrated significantly lower degradation of bothnative LDL (P,0.01) and oxLDL (Figure 5A,P,0.001).Coronary adventitial fibroblasts showed the most avid meta-bolic processing of modified LDL, which exceeded valuesobserved in the coronary and noncoronary SMCs (P,0.001).Differential LDL degradation was not species dependent,because primary cultures of human vascular cells demon-strated a similar pattern (Figure 5B). The avid degradation ofLDL by adventitial cells was maintained even in late passages(passages 2 to 7; not shown).

Expression of SM Differentiation MarkersTo examine whether these characteristics of vascular cells arerelated to their differentiation, several markers of SM differ-entiation were assessed in primary cultures. There waspronounced heterogeneity ofa-SM actin expression in non-coronary SMCs (Table 2). As illustrated in Figure 6,a-SMactin ranged from negative to strongly positive in subconflu-ent cells. The markers of “late” SM differentiation, such as

Figure 4. Synthetic characteristics of vascular cells derivedfrom coronary arteries and noncoronary vascular beds. A, Over-all protein synthesis was examined with 35S-methionine labeling.Comparable protein synthesis was observed in cells derivedfrom aortic media (AO-SMC), iliac artery media (IA-SMC), coro-nary media (CA-SMC), and adventitia (CA-FIB). B, Collagen syn-thesis was measured by 14C-proline labeling followed by colla-genase digestion. Coronary medial SMCs demonstratedsignificantly lower collagen synthesis compared with noncoro-nary medial cells (AO-SMC and IA-SMC) or coronary adventitialcells (CA-FIB). Each column represents mean6SD of 6 to 9 val-ues. *P,0.001 vs all other groups by ANOVA with Bonferroni’scorrection.

Figure 5. LDL degradation in vascular cells derived from por-cine (A) and human (B) coronary and noncoronary vascularbeds. SMCs derived from coronary media (CA-SMC) demon-strated lowest levels of LDL metabolic activities. Noncoronarymedial SMCs showed higher degradation of native LDL (nLDL)and oxLDL compared with coronary medial SMCs, althoughtheir abilities to degrade oxLDL were significantly lower thanthose of adventitial cells. Results are representative of 3 sepa-rate experiments. Each column represents mean6SD of tripli-cate measurements. *P,0.01 vs noncoronary medial SMCs andcoronary fibroblasts; †P,0.01 vs noncoronary SMCs by ANOVAwith Bonferroni’s correction. Abbreviations as in Figure 3.

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h-caldesmon and SM-myosin heavy chain (MHC), also dis-played heterogenous distribution in noncoronary SMCs (Fig-ure 6). Likewise, a similar pattern was observed with SM1-and SM2-MHC antibodies (not shown). In contrast, isolatedcoronary SMCs demonstrated uniform expression ofa-SMactin (Table 2). As shown in Figure 7, they lacked heterog-enous distribution of cytoskeletal markers (a-SM actin,h-caldesmon, and SM-MHC) compared with noncoronarySMCs. This highly differentiated phenotype was present incells cultured in 10% FBS for 1, 3, 5, and 10 days. Figure 7also illustrates the defective spreading of coronary SMCs,which did not change in the presence of several componentsof extracellular matrix (eg, vitronectin, collagen types I andIV, or fibronectin; not shown).

The vast majority of coronary adventitial fibroblasts weredevoid of a-SM actin after isolation (Figure 8). This wasfollowed by a dynamic upregulation ina-SM actin immuno-reactivity in coronary adventitial fibroblasts, which becamealmost uniformly positive fora-SM actin at 5 days, therebyacquiring the characteristics of myofibroblasts.

DiscussionTwo major conclusions can be derived from the results of thepresent study. First, isolated coronary SMCs exhibit a highly

differentiated phenotype, whereas noncoronary SMCs displaygreater phenotypic heterogeneity and more exuberant re-sponses in culture (eg, proliferation, collagen synthesis, LDLdegradation). Second, although coronary SMCs are lessresponsive than other SMCs, coronary adventitial nonmusclecells demonstrate several phenotypic characteristics that en-able them to react to pathophysiological stimuli.

Coronary Medial SMCsDifferent morphology and gene expression, as well as distinctproliferative and migratory capabilities, have been previouslyidentified among noncoronary SMCs.10–12,27–29Whether theselective expansion of certain subtypes of SMCs or theirdifferential survival in culture accounted for phenotypicversatility of noncoronary SMCs remains to be determined.Nevertheless, coronary SMCs, isolated and cultured in anidentical manner, exhibited marked differences. Althoughdifferentiation and growth can be dissociated in culture,30,31

coronary SMCs continued to express differentiation markersand failed to replicate in the presence of several growthfactors (Figure 3). To account for possible species differenc-es,32–34 we also examined human coronary SMCs, whichshowed similar morphology and proliferative activity asporcine coronary SMCs. It should be underscored, however,that our findings do not necessarily contradict previousreports describing the growth ofa-SM actin–positive cellsfrom human coronary arteries.35,36 The particularly greatability of adventitial fibroblasts to migrate, to overgrowmedial SMCs, and to become myofibroblasts (Figure 8) raisesquestions as to the origin of cells previously thought to beSMCs.

The exact mechanism underlying differences betweencoronary and noncoronary SMCs remains to be deter-mined. The development of coronary vessels in situ fromthe coelomic mesothelium, rather than from ectodermal ormesodermal portions of the aorta, may confer different

Figure 6. Photomicrographs illustratingheterogeneity of cytoskeletal markers innoncoronary SMCs. Enzymatically iso-lated porcine aortic medial SMCs weregrown in 10% FBS. They demonstratedheterogeneous distribution of a-SMactin, h-caldesmon, and, to a lesserdegree, SM-MHC (3 to 5 days after plat-ing) (magnification 3490). N/C indicatesnegative control (no primary antibody).

TABLE 2. Percentage of a-SM Actin–Positive Cells in CulturedSMCs Derived From Different Vascular Beds

Time, d AO-SMCs IA-SMCs CA-SMCs

1 7663 2962 9961

3 67614 3565 9961

5 6166 82612 9961

7–10 10060 9662 9961

Enzymatically isolated porcine SMCs (primary cultures) were grown oncoverslips in 10% FBS. The time refers to days in culture after plating.Noncoronary SMCs reached confluence at 7 to 10 days, whereas coronarySMCs remained subconfluent. Results are presented as mean6SD (n510fields, .500 cells counted).

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cellular properties.19,22 In fact, cell type–specific transcrip-tional regulation of differentiation was recently empha-sized by Owens et al.37 The extrinsic signals to beconsidered include extracellular matrix components (eg,laminin, heparan sulfate glycosaminoglycan) that modu-late SMC differentiation.38,39It is less likely, however, thatregional variations in the composition of vascular extra-cellular matrix in situ were responsible for the observeddifferences after enzymatic cell isolation. We also empha-size that the effects of several other factors, which areabsent in culture conditions, may promote dedifferentia-tion of coronary SM in vivo; these factors include age-,gender-, and environment-dependent phenomena (arterialretention of LDL or advanced glycosylation end products),which await clarifications in studies involving the coro-nary vasculature.40 – 42

Coronary Adventitial Nonmuscle CellsThe stimulation of coronary adventitial nonmuscle cellsresulted in phenotypic changes typically attributed to syn-thetic SMCs.43 The expression ofa-SM actin by adventitialnonmuscle cells exemplified the formation of myofibroblasts(Figure 8).44,45 The selective expansion of a small number ofSMCs in adventitial samples was unlikely, inasmuch as

coronary SMCs demonstrated poor attachment and growth(Figures 2 and 3). As shown in the present study, adventitialnonmuscle cells displayed several properties necessary forarterial repair (ie, high adhesion, proliferation, and collagensynthesis). Recently, they have also been shown to possessincreased matrix-degrading activities and migration com-pared with coronary SMCs, which constitutively expressTIMP-1 and -2.46 Notwithstanding these findings, we empha-size that although the conditions for activation of nonmusclecells likely exist after acute mechanical medial injury inhumans,47 the migration of the cells during chronic medialdamage is more difficult to determine. The cytotoxic effectsof modified LDL during atherogenesis,42 however, oftenresult in medial thinning, which may enable nonmuscle celltranslocation and their involvement in lesion formation.48,49

The avid degradation of oxLDL by activated coronary non-muscle cells (Figure 5) suggested a high expression ofscavenger receptor and the possibility of their involvement infoam cell formation on reaching the intima.

Clinical ImplicationsDisappointing results of several clinical studies that targetcoronary restenosis have exemplified a low predictive valueof pharmacological testing in animal models of noncoronary

Figure 7. Photomicrographs demonstrat-ing uniform expression of cytoskeletalmarkers in coronary SMCs. Enzymaticallyisolated porcine coronary SMCs weregrown in 10% FBS. They exhibited uni-form and sustained expression of all SMcell markers (3 to 5 days after plating)(magnification 3490). N/C indicates neg-ative control (mouse IgG instead of pri-mary antibody).

Figure 8. Photomicrographs showingcytoskeletal changes in coronary adventi-tial fibroblasts. Enzymatically isolated por-cine coronary adventitial cells were grownin 10% FBS. They were initially devoid ofa-SM actin but began to acquire a-SMactin in culture (3 to 5 days) and becameuniformly positive for a-SM actin at 10days (magnification 3490).

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arterial injury. The presented results and previous observa-tions in vivo indicate greater susceptibility of coronaryadventitial fibroblasts to mitogenic stimuli compared withcoronary SMCs. Whether the suggested involvement ofadventitial cells will affect the outcomes of antihyperplasticinterventions applied in vivo (eg, brachytherapy or pharma-cological approaches) remains to be determined. Interest-ingly, however, the adventitial delivery of some agentsappears to be more effective than endoluminal administra-tion.50 We postulate that a better understanding of the uniquecharacteristics of coronary vascular cells may provide theinsight necessary for future therapeutic interventions aimedspecifically at a reduction in coronary hyperplastic responses.

In conclusion, in the present study, we examined thecharacteristics of coronary medial SMCs and adventitialnonmuscle cells. Coronary SMCs, which differed from non-coronary SMCs, showed a highly differentiated phenotypewith a limited ability to adhere, proliferate, and synthesizecollagen. In contrast, adventitial nonmuscle cells displayedcharacteristics usually attributed to synthetic SMCs. Wepostulate that the mechanisms of vascular repair and lesionformation may differ among vascular beds, which points tothe importance of future coronary artery–specific investiga-tions in vascular biology.

Note Added in ProofSince the submission of this manuscript, Christen et al51 havealso reported the presence of a highly differentiated pheno-type of coronary SMCs and the unique response of these cellsin culture.

AcknowledgmentsThis study was supported by National Institutes of Health GrantsHL-44150 and HL-60672 and the John S. Sharpe Foundation. Theauthors gratefully acknowledge Kenneth Margulies, MD, and CarolFisher, BS (Temple University), for their assistance in the collectionof human tissues. We thank Mary Ellen Henderson for her assistancein preparation of the manuscript.

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