growth characteristics and cytoskeletal organization of ... · primary stenosing and fresh...

Growth Characteristics and Cytoskeletal Organizationof Cultured Smooth Muscle Cells from Human Primary

Stenosing and Restenosing Lesions

Peter C. Dartsch, Rainer Voisard, Gerhard Bauriedel,Berthold Hofling, and Eberhard Betz

Growth characteristics of human plaque cells selectively extracted from advancedprimary stenosing and fresh restenosing lesions by percutaneous translumlnalatherectomy were studied in vitro. Cells were Isolated either by explant technique orby enzymatic disaggregatlon, and they were Identified as smooth muscle cells (SMC)by positive reaction with antibodies against or-smooth muscle actin. Endothellal cellswere not found In the atherectomlzed tissue. The cells of primary stenosing tissue(ps-SMC) exhibited a significantly low growth rate (0.16±0.04 population doublingsper day) In comparison to the cells of restenosing lesions (re-SMC, 0.64±0.15population doublings per day). Furthermore, ps-SMC became senescent andremained quiescent after two passages, whereas re-SMC retained a high prollferatlveactivity and became quiescent by passage 8 to 10. Both types of cells responded toIncreasing serum concentrations In a dose-dependent manner. Ps-SMC failed torespond to purified platelet-derived growth factor (PDGF) and a mltogen mixtureIsolated from bovine brain (ECGF), but their prollferatlve activity was Increased by theaddition of re-SMC-condltloned culture medium. Despite their high basic growth rate,the prollferatlve activity of re-SMC was significantly stimulated by PDGF and ECGF Ina dose-dependent manner. PS-SMC and re-SMC populations consisted of two distinctsubpopulatlons, which could be discriminated by cell size measurements and celladhesion: 1) relatively small (cell diameter, 18.6±5 /un), low-adhesive, predominantcells, and 2) enlarged (cell diameter, 27.1 ±3 /tun), high-adhesive, flbroblast-llke cellswith abundant mlcrofllaments. Neither ps-SMC or re-SMC stained with antibodiesagainst desmin, but did express vlmentin. The organization patterns of vlmentln andtubulln were unaltered In comparison to each other and to smooth muscle cellscultured from the media of nonatherosclerotic human arteries.(Arteriosclerosis 10:62-75, January/February 1990)

S mooth muscle cell (SMC) proliferation and migrationfrom the arterial media into the subendothelial space

have been recognized as essential components in thedevelopment of atherosclerosis in humans and animals.1-6

The cause for these phenomena are still not completelyunderstood. Although the cellular composition of humanatherosclerotic lesions is well known,1-11 knowledge aboutfunctional, biochemical, and cytoskeletal differencesbetween smooth muscle cells of the normal media andthose of atheromatous plaques is very limited.12-20

Reports on the in vitro growth properties of SMC fromatherosclerotic lesions are still rare. Recently, cell culturehas been used to estimate the growth rate and other

From the Institute of Physiology I, University of Tubingen, Tub-ingen, and the Department of Internal Medicine I, KllnikumGrosshadem, University of Munich, Munich, FRG.

This work was supported by a grant from the Mlnlsterium furWissenschaft und Kunst des Landes Baden-Wurttemberg (FSP26). Rainer Voisard was a recipient of a doctoral fellowship fromBraun Melsungen AG, Melsungen, FRG, and part of this studywas done during his thesis.

Address for reprints: Peter C. Dartsch, Physiological Institute I,University of Tubingen, Gmelinstrasse 5, D-7400 Tubingen 1, FRG.

Received January 2, 1989; revision accepted August 8,1989.

characteristic features of cells isolated from the atheroscle-rotic arteries of animals.21-26 Another approach was that ofGrunwald and Haudenschild26 who showed that initjmalinjury in vivo activates vascular SMC migration and explantoutgrowth in vitro.

In contrast to animal models in which the proliferativeactivity of SMC of experimentally induced atheroscleroticlesions can be studied by a variety of techniques, cellculture is most likely the only way to study human athero-sclerotic material experimentally. There are only a fewreports on cell culture studies of SMC derived from humanatherosclerotic lesions.20'27-30 In these investigations, thegrowth characteristics of cells isolated from atheroscleroticand normal human arteries were compared. There are nocell culture studies of advanced human atheroscleroticlesions or of fresh restenosing lesions at the same location.Such studies may provide a greater insight into the activa-tion process of SMC in vivo.

The present investigation describes the isolation of SMCfrom human primary stenosing lesions (ps-SMC) and reste-nosing lesions (re-SMC) after percutaneous transluminalatherectomy, the cell growth in cultures, and the character-istic features of these cells.

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HUMAN PLAQUE CELLS IN CULTURE Dartsch et al. 63

Table 1. Presentation of All Clinical Data from Patients wtth Advanced Primary Stenoses andFresh Restenoses

Patient Age(yr)

Primary stenosing lesions1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

73

63

45

70

64

48

73

64

61

80

48

63

68

75

64

38

78

89

60

Restenosing lesions

1

2

3

4

5

nd=not c

62

73

52

59

73

tone.

Sex

9 F

3M

S9

3

9

39

36*

3

39

9

39

9

3

3

3S

3

6

9

Stenosis ratedue to

In %

angiographicparameters

Before

95

100

100

100

90

100

100

100

100

80

100

100

90

100

95

100

95

100

70

no data

90

95

80

95

After

15

15

30

30

25

10

30

30

15

30

50

30

20

30

15

25

15

30

20

0

10

35

0

No. of removedspecimens

5

14

7

4

8

3

21

22

7

14

5

11

8

9

2

2

3

3

10

8

6

15

2

6

Total wet weight ofremoved

specimens (mg)

52

184

81

57

94

21

167

347

51

124

43

98

nd

88

102

14

8

33

36

nd

95

79

23

58

MethodsPatients and Plaque Extraction

ArterfosderotJc plaque material was selectively extractedfrom severely stenosed or completely occluded superfi-cial femoral arteries by the Simpson atherectomy deviceas previously described.3132 The selective capture ofstenosing tissue was achieved by adjunctive angioscopy.33

A total of 158 specimens from primary stenosing lesions from19 patients (ages 64±12 years) and 37 specimens fromrestenosing lesions of five patients (ages 64 ±9 years) wereobtained at the Department of Internal Medicine I, KlinikumGrosshadern, University of Munich, FRG. Atherectomyspecimens were usually 2 to 8 mm long and about 1 mmthick. The total wet weight of primary stenosing tissue ofall patients was 1600 mg (average, 89 mg/patient) and255 mg for tissue of restenosing lesions (average, 64 mg/patient). The average stenosis rate of all patients due toangiographic parameters before percutaneous atherec-tomy was 95%±8%, and the residual stenosis rate afterintervention was 22%±12%, thus demonstrating that themain portion of stenosing tissue was extracted. For adetailed presentation of the clinical data, see Table 1.

Cell Isolation and CultivationImmediately after extraction, plaque specimens were

transferred to glass flasks containing sterile and HEPES-buffered (15 mM) culture medium without serum supple-ment. The specimens were transported within 20 to24 hours to the Physiological Institute I, University ofTubingen, Tubingen, FRG, for cell isolation and cultiva-tion (by permission of the ethical committee of theUniversity of Tubingen). Here, specimens were washedtwice with fresh medium (see above). One third of theplaque cylinders were cut into small pieces of about1x1 mm and were cultivated as adherent explants.3436

The remaining specimens were enzymatically disaggre-gated for 180 minutes at 37°C in a shaking water bath bythe following enzyme mixture: 10 ml of HEPES-bufferedculture medium containing 18 mg collagenase (Worthing-ton CLS III, 229 lU/mg, charge 45S 8973; Seromed,Berlin, FRG), 2 mg elastase (from porcine pancreas,charge 11569820-05; Boehringer-Mannheim, Mannheim,FRG), and 10 mg trypsin inhibitor (from soybean,53.5 lU/mg; Serva, Heidelberg, FRG), pH 7.2. After theaddition of 20% fetal calf serum, the cells were centri-



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64 ARTERIOSCLEROSIS VOL 10, No 1, JANUARY/FEBRUARY 1990

fuged for 10 minutes at 170 g and were plated. The cellswere then cultured in a mixture of Waymouth's MB 752/1and Ham F12 nutrient mixture (1=1; vol/vol) supple-mented with 10% to 15% fetal calf serum at 37°C in 7%CO2. All cell culture reagents were purchased from GibcoBRL, Eggenstein, FRG. At confluency, cells were subcul-tured by trypsin/ethylenediaminetetraacetic acid (EDTA)treatment (0.05%, pH 7.2). For additional information oncell culture techniques used for the cultivation of SMCfrom human stenosing tissue as extracted by percutane-ous atherectomy, see reference 33.

Immunofluorescence MicroscopyFor cell identification and examination of cytoskeletal

components, the cells of the primary cultures and firstsubcultures were seeded on round glass coverslips at adensity of 1000 to 5000 cells/cm2. At 48 hours after thecells were completely spread out, they were fixed inmethanol for 6 minutes at -20°C, and indirect immune-fluorescence was carried out as described.3837 The fol-lowing primary antibodies were used at a concentrationof 50 ^ig/ml: 1) monoclonal anti-a-smooth muscle actin(Progen Biotechnik, Heidelberg, FRG), 2) monoclonalanti-a-tubulin (Amersham Buchler, Braunschweig, FRG),3) monoclonal anti-human desmin (Dakopatts, Glostrup,Denmark); 4) monoclonal anti-vimentin (a kind gift ofMary Osbom, G6ttingen, FRG), and 5) monoclonal anti-factor Vlll-related antigen (vWF; Calbiochem, Frankfurt,FRG). Tetrareodaminyl-isothiocyanate (TRiTC)- andfluorescein-isothyocyanate (FITC)-labeled second anti-bodies (goat antimouse IgG) were purchased from MilesScientific, Munich, FRG, and Dianova, Hamburg, FRG.Counterstaining of the nuclei was performed with DAPI(4',6-diamidino-2-phenylindole-dihydrochloride; ServaFeinbiochemika, Heidelberg, FRG) at a concentration of0.1 MO/ml in phosphate-buffered saline (PBS) for 20 min-utes at 37°C. Staining of stress fibers was carried out withTRITC-phalloidin as previously described.38 The cellswere mounted in Moviol 4-8837 and examined using aNikon Optiphot microscope equipped for epifluores-cence with appropriate filter sets. Micrographs weretaken with a Planapo 40/1.0 Oil or a Planapo 60/1.4 Oillens on Kodak Tri-X Pan film.

Examination of Cell ViabilityCell viability was checked by using fluorescent dyes as

follows38: Cell cultures were washed twice with PBS andincubated for 2 minutes at room temperature with PBScontaining 6 Mg/ml fluorescein diacetate and 3 /xg/mlethidium bromide (both compounds were purchasedfrom Sigma Chemie, Deisenhofen, FRG). After removal ofdyes and another two washes with PBS, the cells wereexamined with an inverted microscope (Nikon DiaphotTMD) by using epifluorescence. Viable cells that canhydrolyse fluorescein diacetate exhibit a green fluores-cent staining of cytoplasm when blue excitation is used,whereas the nuclei of dead cells show a red fluorescentstaining when green excitation is used.

Growth CurvesStudies on cell proliferation were not possible in pri-

mary cultures because the number of enzymatically iso-lated cells was not high enough for a series of growthcurves. Therefore, ps-SC and re-SMC of confluent pri-mary cultures were trypsinized and seeded into 6-wellplates (Costar 3406, Tecnomara, Fernwald, FRG) at adensity of 2000 to 3000 cells/cm2. At 24 hours afterseeding and on every third day thereafter, the mediumwas exchanged. At appropriate times, cells of two wellswere washed with PBS, were trypsinized for 10 minutes at37°C, and were transferred to a cell counter (CASY 1,Scharfe Systems, Reitiingen, FRG) for measurement ofcell number and cell size. Both parameters were moni-tored up to 16 days after seeding.

Growth Factors and Collectionof Conditioned Medium

Platelet-derived growth factor (PDGF, purity>95%) fromhuman platelets was purchased from Flow Laboratories,Meckenheim, FRG (charges 87-1110 and 88-5045). Thiswas diluted with PBS containing 2 mg/ml bovine serumalbumin and was used at final concentrations rangingfrom 1 to 5 ng/ml. According to the method described bySavoly et al.,40 a mitogen mixture (ECGF) was isolatedfrom bovine brain and used at final concentrations rang-ing from 25 to 100 /xg/ml. Re-SMC conditioned mediumwas removed from cultures in log-phase under sterileconditions and immediately frozen and stored at - 7 0 X .

Collagen Coating of Petri DishesLathyritic collagen type I was purchased from Boeh-

ringer-Mannheim, Mannheim, FRG, and was diluted with0.1 M acetic acid to a final concentration of 0.1 mg/ml.This solution was pipetted into Petri dishes, which werethen incubated for 30 minutes at 37°C. After the removalof collagen solution, the dishes were dried for another30 minutes under ultraviolet light and were immediatelytaken for the experiments.

Cell Adhesion AssayThe assay for cell adhesion was a slight modification of

that described by Bjdrkerud.41 The cultures of ps-SMCand re-SMC from which the cells were to be assayedwere detached to a single-cell suspension by trypsiniza-tion, were diluted with culture medium, and were centri-fuged at 170 ^ fo r 7 minutes. Pelleted cells were resus-pended with Waymouth's MB 752/1 and Ham F12 (1 = 1)containing 1 mg/ml bovine serum albumin (Merck, Darm-stadt, FRG) and standard amounts of antibiotics (the"adhesion medium") and were washed twice with thismedium by centrifugation. Finally, the cells were resus-pended in adhesion medium, counted, and seeded inaliquots of 5 ml into tissue culture plastic dishes (Falcon3002F). The dishes were either untreated or coated withlathyritic collagen type I. After cell seeding, the disheswere incubated at 37°C in the incubator. At appropriatetimes, the supernatant was removed, and the dishes werecarefully washed twice with PBS. Adhering cells were



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HUMAN PLAQUE CELLS IN CULTURE Dartsch et al. m

BFigure 1 . A. High-magnification photomicrograph of a smoothmuscle cell from advanced primary stenosing lesions (ps-SMC)in the first subculture with abundant stress fibers in cytoplasmand a granulated surface. B. Actively migrating smooth musclecell from restenosing lesions (re-SMC) in the first subcultureshowing neither a granulated surface nor a pronounced networkof stress fibers. Bar=50 juri.

detached by trypsin/EDTA, and cell numbers and cell sizewere measured in the cell counter described above.

ResultsCharacteristic Features of Cells In PrimaryCultures and Subcultures

Within 4 to 8 days after adhesion of tissue explants, thecells of primary stenosing tissue started to grow outradially from approximately 70% of the explant pieces.These cells were identified as SMC (see next section).The vast majority of ps-SMC exhibited abundant stressfibers running through the cytoplasm (Figure 1A). Enzy-matically obtained ps-SMC attached 24 to 48 hours afterplating and exhibited the same characteristics as cellsthat had grown out of the explants. For a detaileddescription of ps-SMC morphology, see references 33and 42. We were able to subcultivate ps-SMC two times.Thereafter, the cells remained viable, but did not undergofurther cell divisions. Another trypsin treatment of the cell

cultures for subculturing to induce further cell divisions43

resulted in the death of ps-SMC.In contrast, re-SMC started to migrate and proliferate

from the explants after 2 to 3 days, and about 90% of theadherent explants exhibited an abundant outgrowth ofre-SMC. Re-SMC obtained by enzymatic disaggregationstarted to proliferate immediately after attachment of cellsplated as primary cultures and exhibited an extraordinar-ily high proliferation rate, as calculated by daily countingthe number of cells in at least five arbitrarily selectedmicroscopic fields.

Most re-SMC were elongated and fibroblast-like, butpolygonally shaped cells were also observed. In contrastto ps-SMC, cells from restenosing tissue did not showextraordinarily pronounced stress fibers in cytoplasm(Figure 1B). The cells of primary cultures were subculti-vated after reaching confluency and then routinely aftertwo population doublings per passage. Signs of senes-cence, such as a decreased capacity for further dou-blings or abundant cell debris,44 were observed after fivepassages. By passage 8 to 10, re-SMC became quies-cent and did not undergo further cell divisions.

Common to both ps-SMC and re-SMC was the rela-tively small number of cells that could be isolated byenzymatic disaggregation of plaque tissue. The yield ofSMC from primary stenosing and restenosing lesionsvaried largely, but was in all cases relatively small (approx-imately 30 000 cells/100 mg of stenosing tissue). This factis not surprising since atherectomized tissue of lesions ofthe superficial femoral artery contains abundant amountsof extracellular matrix material but only a relatively smallnumber of SMC (unpublished observations).

Identification of Isolated Cells andCytoskeletal Organization

Isolated cells were identified in primary cultures andfirst subcultures by their positive reaction with antibodiesagainst a-smooth muscle actin, which has been shown tobe a specific marker for SMC46 (Figures 2A and 2B). Thestaining with antibodies against a-smooth muscle actindid not differ from the one achieved by TRITC-phalloidin(not depicted), suggesting a distribution of a-actin alongthe whole stress fiber system. The polygonally shapedcells also reacted strongly with anti-a-smooth muscleactin, characterizing these cells, in spite of their strangecell shape, as SMC. According to these immunologicalcriteria, at least 90% of ps-SMC and re-SMC in primarycultures were SMC. A positive reaction with antibodiesagainst vWF, a marker for endothelial cells,4847'48 was notobserved.

To examine whether there were significant differencesin cytoskeletal organization between ps-SMC and re-SMC, we checked the staining patterns of tubulin andintermediate-sized filaments by indirect immunofluores-cence microscopy and by using specific antibodies.Microtubules in ps-SMC and re-SMC originated at areasadjacent to the nuclei (microtubule organizing centers),ran radially through the cytoplasm, and terminated at thecell periphery (Figure 2C).

Vimentin was observed to be organized as wavy fibersextending through the cytoplasm in a more or less radial



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Figure 2. Cytoskeletal organization of cells from human stenoslng tissue. A, B. Positive reaction with antibodies against o-smoothmuscle actin showing long and nonlnteraipted filaments. C. Network of mtarotubules originating at the microtubule organizing centersaround the nucleus and terminating near the cell periphery. D. Pattern of distribution of vimentin showing wavy fibers extending throughthe cytoplasm. Cells failed to react with antibodies against desmin. Bars=25 ̂ m.

arrangement and as a particularly abundant networkaround the nucleus (Figure 2D) as described for animalcells (see references 49 and 50). There was no differencein the microhjbule staining pattern between ps-SMC andre-SMC nor |n vimentin organization in comparison to SMCcytoskeietons obtained from cells of nonatherosderotjchuman artery walls (not depicted). No positive reaction ofps-SMC and re-SMC in primary cultures and subcultureswith antibodies against desmin was observed. These datacomplement and extend those of others1751S2S3 whoreported that cells that have migrated from the media intothe intima during atherogenesis contain decreased, if any,amounts of desmin and increased amounts of vimentin.

Cell Size Measurements and Growth CurvesMeasurements of cell size of total ps-SMC and re-SMC

populations in primary cultures and first subculturesrevealed a pronounced heterogeneity (Figures 3A and3B). Both SMC populations from human atheroscleroticlesions consisted of two distinct subpopulations, whichcould be easily distinguished by cell diameter: 1) sub-population 1 contained cells with a cell diameter of

18.6±5 fim (mean±SD), and 2) subpopulation 2 con-tained cells with a diameter of 27.1 ±3 nm (mean±SD).The cell diameters of the two subpopulations differedsignificantly (/O<0.01, Student's /test). However, ps-SMCand re-SMC populations showed a clear predominanceof cells with the smaller diameter: 70% of total cellnumber of ps-SMC and 85% of re-SMC were designatedas subpopulation 1. In contrast, cells of subpopulation 2,which might represent the enlarged and senescent por-tion of cells, were present at 30% for ps-SMC and only at15% for re-SMC. Interestingly, the amount of debrispresent in the cultures was extremely high for ps-SMCcultures (45% of total counts in cell counter), whereas inre-SMC cultures, the amount of debris was only 15% oftotal counts in cell counter. As shown in Figure 3C forre-SMC of the fifth subculture, the amount of cell debrisand the portion of enlarged cells of subpopulation 2increased with in vitro age, but did not attain the valuesestimated for ps-SMC in first subcultures.

Since the cell yield after enzymatic disaggregation wasnot sufficient for a series of growth curves, detailedstudies on cell proliferation were not possible on primary



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SMOOTH MUSCLE CELLS FROM ADVANCED PRIHARVSTENOSIHG LESIONS IN FIRST SUBCULTURE

HESSVOLUHEH:

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COUNTS t 49783COUNTS/nll 28742

COUNTS/MESSUNG:MESSUHG II 8866HESSUNG 2: 7995MESSUNG 31 8886MESSUNG 4: 8583HESSUNG 5i 8585MESSUNG 6: 8628MITTELMERT:8297

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Figure 3. The original plots of cell size of counter of A. primary stenosing lesion smooth muscle cells (ps-SMC) and B. restenosing lesionsmooth muscle cells (re-SMC) in the first subcultures (in vitro age of five cumulative population doublings). The cell populations of both originsconsist of two distinct subpopuiations with different cell diameters, which can be quantified by cursor settings: subpopuiation 1 (SP-1) withcell diameters of 18.6±5 tan (mean±SD) and subpopuiation 2 (SP-2) with cell diameters of 27.1+3 nm (mean±SD). Note the clearpredominance of the smaller cells in both cell populations and the different amounts of debris. C. Cell size plot of re-SMC in the fifth subculture(approximately 15 population doublings) showing the increase of cellular debris, but still only a small portion of enlarged cells. For a betterunderstanding of the cell size plots, the statistical analyses (also printed by the counter) were removed.



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Table 2. Presentation of All Experimental Data in This Study

PatientOutgrowth fromexplant pieces*

Primary stenosing lesions

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Restenosing

1

2

3

4

5

-4-

nd

+ +++nd

±

++++nd

++ +

+

+lesions

nd

+ + +

+ + +

nd

+ + +

Cell attachment andvitality after enzymatic

disaggregation*

-

++

+ +nd

++

++ +++

+ +++

+ +

+++

+ + ++ + +

+ + +

++ + +

Growth rates (populationdoublings/day) in passage

cni

cni

cni

0.188 in passage 1

0.100 in passage 1

cni

cni

cni

cni

0.147 in passage 1

cni

cni

0.125 in primary culture

cni

cni

0.204 in passage 1

0.187 in passage 2

cni

cni

cni

0.500 in primary culture!

0.525 in passage 1

0.493 in passage 2

0.245 in passage 6

0.750 in passage 2

0.798 in passage 3

cni

nd

nd=not done; cni=cell number insufficient.*Evaluations from excellent (+ + +) to poor (-).•Calculated by counting the cell number of at least five arbitrarily selected microscopic fields of the inverted

microscope.

cultures. For an examination of growth rates (Table 2),only plaque cells that were isolated by enzymatic disag-gregation were taken for these studies. This enabled usto define the in vitro age accurately. Despite their differentgrowth rates (see below), only cells with an in vitro age offive cumulative population doublings were used fordetailed measurements of growth rates in the first sub-cultures. The growth curves of total ps-SMC and re-SMCpopulations in the first subcultures showed that theproliferative activity of re-SMC was extraordinarily high incomparison to ps-SMC (Figure 4). This demonstrates thatthe metabolic activity of SMC in fresh human restenosinglesions is higher than that of primary stenoses. The meanvalue±standard deviation of all growth curves of ps-SMCwas 0.16±0.04, whereas that of re-SMC was 0.64±0.15.The difference was statistically significant (/CxO.001).Detailed growth curves of re-SMC removed by percuta-neous atherectomy were possible with restenosing plaquematerial from two different patients (Table 2). In addition,

the growth rates of re-SMC from a third patient could becalculated by counting the number of cells of at least fivearbitrarily selected microscopic fields. On the other hand,another three detailed proliferation studies on restenos-ing lesions, which were extracted by surgical interventionfrom peripheral and coronary arteries (unpublished results),showed the same increased growth rates of re-SMC inculture. Additional proliferation studies on re-SMC in thethird and sixth passages also showed an increased growthrate in comparison to ps-SMC (Table 2), indicating that theactivation of SMC in vivo in fresh restenosing lesions isretained over several passages in vitro.

At first glance, the increased growth rates of re-SMCseemed to be related mainly to the smaller cells ofsubpopulation 1 (Figure 5B). A careful calculation ofsubpopulation doubling rates showed instead that theproliferative activity of cells of both subpopulations wasstimulated at the same degree. Despite this fact, the cellsof subpopulation 1 predominated in all experiments



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Table 2. Presentation of All Experimental Data In This Study

PatientOutgrowth fromexplant pieces*

Primary stenosing lesions

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Restenosing

1

2

3

4

5

±

nd

+ +++nd

±

++++nd

++ +

+±

+lesions

nd

+ + +

+ + +

nd

+ + +

Cell attachment andvitality after enzymatic

disaggregation*

-

++

+ +nd++

++ +++

+ +++

+ +

+++

+ + ++ + +

+ + +

++ + +

Growth rates (populationdoublings/day) in passage

cnl

cni

cni

0.188 in passage 1

0.100 in passage 1

cni

cni

cnl

cni

0.147 in passage 1

cni

cni

0.125 in primary culture

cnl

cni

0.204 in passage 1

0.187 in passage 2

cni

cni

cnl

0.500 in primary culture!

0.525 In passage 1

0.493 In passage 2

0.245 In passage 6

0.750 in passage 2

0.798 in passage 3

cni

nd

nd=not done; cnl=cell number Insufficient.•Evaluations from excellent (+ + +) to poor (-).•Calculated by counting the cell number of at least five arbitrarily selected microscopic fields of the Inverted

microscope.

cultures. For an examination of growth rates (Table 2),only plaque cells that were isolated by enzymatic disag-gregation were taken for these studies. This enabled usto define the in vitro age accurately. Despite their differentgrowth rates (see below), only cells with an in vitro age offive cumulative population doublings were used fordetailed measurements of growth rates in the first sub-cultures. The growth curves of total ps-SMC and re-SMCpopulations in the first subcultures showed that theproliferative activity of re-SMC was extraordinarily high incomparison to ps-SMC (Figure 4). This demonstrates thatthe metabolic activity of SMC in fresh human restenosinglesions is higher than that of primary stenoses. The meanvalue±standard deviation of all growth curves of ps-SMCwas 0.16±0.04, whereas that of re-SMC was 0.64±0.15.The difference was statistically significant (/CxO.001).Detailed growth curves of re-SMC removed by percuta-neous atherectomy were possible with restenosing plaquematerial from two different patients (Table 2). In addition,

the growth rates of re-SMC from a third patient could becalculated by counting the number of cells of at least fivearbitrarily selected microscopic fields. On the other hand,another three detailed proliferation studies on restenos-ing lesions, which were extracted by surgical interventionfrom peripheral and coronary arteries (unpublished results),showed the same increased growth rates of re-SMC inculture. Additional proliferation studies on re-SMC in thethird and sixth passages also showed an increased growthrate in comparison to ps-SMC (Table 2), indicating that theactivation of SMC in vivo in fresh restenosing lesions isretained over several passages in vitro.

At first glance, the increased growth rates of re-SMCseemed to be related mainly to the smaller cells ofsubpopulation 1 (Figure 5B). A careful calculation ofsubpopulation doubling rates showed instead that theproliferative activity of cells of both subpopulatjons wasstimulated at the same degree. Despite this fact, the cellsof subpopulation 1 predominated in all experiments



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Days in cultureFigure 4. Representative growth curves of the total cell popu-lations of primary stenosing lesion smooth muscle cells(ps-SMC) (o —o) versus restenosing lesion smooth muscle cells(re-SMC) ( • — • ) . The smooth muscle cells were examined in thefirst subculture (in vitro age of five cumulative population dou-blings). Their proliferation was examined In the presence of15% fetal calf serum. Re-SMC exhibited significantly highergrowth rates than did ps-SMC. The data are expressed as meanvalues±standard deviations.

because the smaller cells became stationary at highercell densities than those of subpopulation 2. In compar-ison, both ps-SMC subpopulations exhibited a less pro-nounced prolrferative activity, resulting in an overallreduced growth rate (Figure 5A).

Response of Cells to Serum Mftogens andGrowth Factors

Plaque-derived cells were routinely cultivated in culturemedium supplemented with 10% to 15% fetal calf serum.To examine if the proliferative activity of the cells can beinfluenced by varying serum concentrations and thusdifferent amounts of serum mitogens, ps-SMC andre-SMC were cultured in medium containing serum con-centrations ranging from 1 % to 20%. Despite their signif-icantly low growth rates in culture, ps-SMC clearlyresponded to increasing serum concentrations in a dose-dependent manner (Figure 6A). Serum concentrations of1% and 5% did not stimulate proliferative activity ofps-SMC, and the cells remained quiescent. The prolifer-ative activity of re-SMC was also stimulated by increasingserum concentrations in a dose-dependent manner (Fig-ure 6B). In contrast to ps-SMC, re-SMC that were exposedto a serum concentration of 5% did not remain quiescent,

Days In culture Days In culture

Figure 5. Detailed presentation of the proliferative activity ofcells of the two different subpopulations that were termed SP-1( • - • ) and SP-2 ( 0 - 0 ) and differed significantly from eachother In their cell diameters as shown in Figure 3. A. Cellproliferation of primary stenosing lesion smooth muscle cell(ps-SMC) subpopulations. B. Cell proliferation of restenosingsmooth muscle cell (re-SMC) subpopulations. Note the predom-inance of the smaller cells of SP-1, especially in the re-SMCpopulation. The data are expressed as mean values±standarddeviations.

indicating again the activated state of cells from freshrestenosing lesions. The use of pooled and heat-inactivated (30 minutes at 56°C) homologous whole bloodserum from three healthy donors did not increase thegrowth rates of either ps-SMC or re-SMC when comparedwith the corresponding concentrations of fetal calf serum(not depicted).

Surprisingly, as shown in Figure 7, ps-SMC did notrespond to the increasing PDGF and ECGF concentra-tions present in culture medium. In contrast, the prolifer-ative activity of re-SMC was stimulated by both growthfactors in a dose-dependent manner. Under our cellculture conditions and the growth factor concentrationsused in the test assays, re-SMC proliferation could bestimulated more than 2.5-fold in comparison to the cor-responding controls.

Figure 6. Growth curves of primary stenosing lesion smoothmuscle cells (A) and restenosing lesion smooth muscle cells (B)in response to serum concentrations ranging from 1% to 20%.Note that growth rates of cells of both origins were increased ina dose-dependent manner.



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iRgure 7. Relative cell proliferation of primary stenosing lesion smooth muscle cells (ps-SMC) (A, C)and restenosing lesion smooth muscle cells (re-SMC) (B, D) after 7 days of continuous incubation withplatelet-derived growth factor (PDQF) concentrations ranging from 1 to 5 ng/ml (A, B) and endothelialcell growth factor (ECGF) concentrations ranging from 25 to 100 /ifl/ml (C, D). Note that ps-SMC didnot respond to growth factors, whereas the proliferative activity of re-SMC was stimulated in adose-dependent manner. The data are expressed as mean values±standard deviations of oneexperiment with pooled cells from two patients in each case.

/response of Ps-SMC to Re-SMC ConditionedCulture Medium

Since it was shown that cells derived from humanatheromas release a PDGF-like mitogenic activity into theculture medium,54 we examined the effect of re-SMCconditioned medium on the growth rates of cells fromadvanced primary stenosing lesions. As shown in Fig-ure 8, the addition of re-SMC conditioned cultured mediumcaused a significant increase of ps-SMC proliferation incomparison to controls. The effect was not visible atonce, but took at least 2 days until ps-SMC proliferationwas stimulated. In the experiments, ps-SMC, which weregrown in medium with the corresponding concentration(15%) of fetal calf serum, served as controls.

Differential Adhesion ofRe-SMC Subpopulatlons

To further characterize the re-SMC subpopulations, anattachment assay was performed according to the methodof Bjorkerud.41 As shown in Figure 9, the enlarged cells ofsubpopulation 2 were high adhesive, whereas the smallercells of subpopulation 1 were low adhesive. At as soon as30 minutes after seeding, the vast majority of subpopula-tion 2 cells were attached, but only a small portion ofsubpopulation 1 cells were attached. The smaller cellswere attached only after a 60-minute incubation. Thisdifferential adhesion of the two subpopulations may beused for separation and independent growth in cellcultures. The attachment of both re-SMC subpopulationson collagen type I, which promotes adhesion of SMCfrom nonatherosclerotic human arteries (unpublishedobservations), was delayed.

Influence of Substrate Coating onRe-SMC Proliferation.

The results of the adhesion assay demonstrated thatcollagen type I delayed the adhesion of both re-SMCsubpopulations. To determine whether collagen type I alsodecreases the proliferative activity of re-SMC, an additionalproliferation assay was carried out. The results show thatcollagen type I inhibited re-SMC proliferation in compari-son to tissue culture plastic (Figure 10A). The proliferativeactivity of SMC from nonatherosclerotic human arterieswas not influenced by collagen type I (Figure 10B).

DiscussionPercutaneous atherectomy with the Simpson catheter

recanalizes severely stenosed or even completelyoccluded arteries in humans.3132556657 This clinicallyvaluable device makes it possible to collect stenosingtissue removed in the tubular housing and thus to studyextracted plaque material by a variety of techniques, suchas histology,32 immunohistochemistry (Dartsch et al.,unpublished observations), gel electrophoresis, and cellculture.3342 In this article, the isolation and cultivation ofSMC from advanced human primary stenosing lesionsand from fresh restenosing lesions after removal by theSimpson atherectomy device is described. Moreover, thegrowth characteristics and cytoskeletal organization ofcells from both sources, as well as their different reactionpatterns in cell culture, are compared.

Common to both ps-SMC and re-SMC was the clearsubdivision of total cell populations into two subpopula-tions with different cell diameters. Concerning cell shape,



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Days in cultureFigure 8. Exposure of primary stenosing lesion smooth mus-cle cells to restenosing lesion smooth muscle cell conditionedmedium ( • - • ) caused an increased prollferative activity. • - •is the corresponding control wtth 15% fetal calf serum. Thearrow indicates the addition of conditioned culture medium.Data are expressed as mean values+standard deviations.

a cellular heterogeneity In human atherosclerotic lesionshas already been described by various authors11-S8B9 andhas been addressed as a cellular polymorphism.686061

Only Bjorkerud30'4162-63'64 has found two distinct subpop-ulatjons in the cultures of SMC derived from human andanimal arteries. Bjorkerud also demonstrated that thesmall cells were low adhesive (I cells), whereas the largercells were high adhesive (A cells). He suggested that thedifferent types of arterial SMC might reflect specializedsubpopulations, with the I cells possibly involved in repairand remodelling of the artery.

As we have seen from our studies, the overall predom-inance of the smaller cells of subpopulation 1 in ps-SMCand re-SMC total populations is suggestive that thishighly activated SMC subpopulation may be the cell typethat has originally migrated from the media into thesubendothelial space during the initial stages of plaquedevelopment during atherogenesis. Moreover, the twosubpopulations discriminated in our study could be char-acterized by differential adhesion after seeding with thesmaller cells of subpopulation 1 as low adhesive and theenlarged cells of subpopulation 2 as high adhesive cells.From all parameters described, these two subpopula-tions of our study seem to be identical with those thathave already been described by Bjorkerud, although inour adhesion assay, the cells attached to the substrateconsiderably faster. The delayed cell attachment anddecreased proliferation of both re-SMC subpopulationson collagen type I in comparison to tissue culture plasticmay be due to the fact that cells in human fibromuscular

intjmal thickenings preferentially synthesize collagen type IIIand only small amounts of collagen type I.65

Another major finding of this study is that cultured SMCfrom fresh restenosing lesions grew at a significantlyhigher rate than cells from advanced primary stenosinglesions. Our results demonstrate that ps-SMC exhibitedan extraordinarily low growth rate in primary cultures andbecame quiescent after being subcultured once or twice.In addition, ps-SMC behaved like senescent cells, notonly in their proliferative activity and cellular debris, butalso in revealing an abundant microfilament network withcytoplasmic vacuoles and a granulated surface. In con-trast, cells from fresh restenosing lesions did not possessthese criteria of senescence, even when subcultivatedseveral times. Since SMC derived from nonatheroscle-rotic arteries have decreased growth rates and growthcapacities with increasing donor age (unpublished obser-vations), one could conclude that there may be aninfluence of donor age in the present study. As shown indetail (Table 1), the two patient populations (with primarystenoses and restenoses) did not differ, so that thispossible influence can be excluded in the evaluation ofgrowth rates.

Re-SMC showed an extraordinarily high growth rate inall proliferation studies, with the cells in the first andsecond subcultures indicating the cellular activation invivo. This increased proliferative activity of SMC, which isonly one facet of an altered complex metabolic situation,was also observed in specimens from one patient wherethe medial layer was injured during atherectomy interven-tion. These observations agree with the findings of Grun-wald and Haudenschild26 on cultured SMC from balloon-injured rat aortas, which grew more rapidly in vitro andeven became temporarily serum independent. Severalother reports have also demonstrated that SMC isolatedfrom early lesions, as presented in this study by freshrestenosing tissue, from both animals2366 and humans,29

proliferate at a higher rate than do cells from moreadvanced lesions or from the surrounding media.

Since the number of cumulative population doublingsof ps-SMC and re-SMC in proliferation studies with cellsof first subculture was identical, the early senescence ofps-SMC in culture can only be explained by the fact thatps-SMC must have undergone more cell doublings dur-ing atherogenesis in vivo. From our immunohis-tochemical studies on human plaque material removedfrom the superficial femoral artery, we know that suchplaques contain abundant amounts of extracellular matrixmaterial, but only a relatively small number of SMC, whichare mainly distributed in the form of cell clusters (Dartschet al., unpublished observations). When considering thecell yield of approximately 30 000 cells/100 mg of stenosingtissue and the lifespan of re-SMC in culture of about 10 celldoublings more than ps-SMC, one may conclude thatonly a very limited number of SMC must have originallymigrated from the media into the subendothelial space.

The in vitro growth characteristics of SMC derived fromhuman atherosclerotic lesions have already beenevaluated.27-3061 •*" The results of these studies are con-tradictory, because a reduced proliferative activity ofplaque cells27 as well as an unaltered283061 or even



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ADHESION OP SMOOTH MUSOJ CH1S FROMHUMANRESTEI4OSMG LESIONS ON COLLAGEN TYPE I

M M . AFTER SEEOMG)

ADHESION OP SMOOTH MUSCLE CELLS PROM HUMANRESTENOSMG LESIONS ON TISSUE CULTURE PLASTIC

(30 M M . AFTER SEEDING)

B ADHESION OP SMOOTH MUSCLE CELLS FROM HUMANRESTENOSMG LESIONS ON COLLAGEN TYPE I

(60 M M . AFTER SEEDNO)CLI*CII4M

ADHESION OF SMOOTH MUSCLE CELLS FROM HUMANRESTENOSMG LESIONS ON TISSUE CULTURE PLASTIC

(60 M M . AFTER SEEDMG)

Figure 9. Differential adhesion of restenosing lesion smooth muscle cell (re-SMC) subpopuiations 30 minutes (A, C) and 60 minutes(B, D) after seeding. The figure shows the amount of attached cells. The enlarged cells of subpopulatlon 2 (SP-2) attached earlier thanthe smaller cells of subpopulation 1 (SP-1). The attachment of both re-SMC subpopuiations was delayed by coating Petri dishes withcollagen type I (A, B) In comparison to tissue culture plastic (C, D).

increased29 cell proliferation rate of cells from atheroscle-rotic lesions in comparison to unaffected arterial mediawere reported. These contradictions may be partiallycaused by different plaque locations (for example, aorta,femoral artery) and by differences in cell culture tech-niques. In a series of experiments, Ross et al.27 havecompared the proliferative response of SMC obtainedfrom advanced lesions of the superficial femoral arterywith those obtained from the underlying media of thesame artery. They used increasing concentrations ofhomologous whole blood serum and observed that theSMC from the lesions failed to respond to concentrationsof serum ranging from 1 % to 10% at either passage. Theytermed these low responders "senescent cells." In con-trast, our observations demonstrate that both ps-SMCand re-SMC responded to increasing serum concentra-tions in a dose-dependent manner with an increase inproliferative activity. The only difference between ps-SMCand re-SMC was that cells from primary stenosing lesionsremained quiescent at low serum concentrations up to5%, whereas the growth rates of cells from restenosinglesions could be stimulated by such low concentrations.

Although ps-SMC clearly responded to increasingamounts of serum mitogens, they failed to respond topurified PDGF or a mitogenic mixture, ECGF, whilere-SMC were again stimulated by both growth factors in adose-dependent manner. On the other hand, the use ofre-SMC conditioned culture medium enhanced growthrates of ps-SMC. An interpretation of all these observa-tions is difficult. The highly activated cells from restenos-ing lesions obviously produce a mitogenic activity, whichmay stimulate their own growth in an autocrine manner68

and which is released into the culture medium. Thismitogenic activity probably differs from PDGF, as purifiedPDGF from human platelets did not stimulate growthrates of ps-SMC. Walker et al.6 9 7 0 have reported thatSMC isolated from arterial intima 2 weeks after a ballooncatheter-induced injury produced 10 times as muchPDGF-like material as SMC isolated from the media ofuninjured arteries. Furthermore, Morisaki et al.71 haveshown that SMC secrete a growth factor that is distinctfrom PDGF. We propose the following explanation for ourfindings: Cells from early lesions, as represented by freshrestenosing lesions, respond to PDGF by proliferating at



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Days in cultureFigure 10. The Influence of substrate coating on the prolifera-tion of restenosing lesion smooth muscle cells (re-SMC) (A) andsmooth muscle cells from nonatherosclerotic human arteries(B). Re-SMC proliferation was decreased by collagen type I(A —A) when compared with tissue culture plastic ( • - • ) . Theproliferative activity of smooth muscle cells from nonatheroscle-rotic human arteries was not influenced by coating Petri disheswith collagen type I. The data are expressed as meanvalues ±standard deviations of one experiment with pooled cellsfrom two donors in each case.

a higher rate. After several cell doublings in vivo, plaquecells no longer respond to PDGF, and the process offurther plaque development by SMC proliferation is stim-ulated by the cells actively secreting a self-stimulating(autocrine) growth factor that differs from PDGF. Thishypothesis is supported by the results of Libby et al.64

who demonstrated that SMC from diseased human arter-ies can secrete mrtogenic activity, from which only about38% could be neutralized by antj-PDGF antibodies.Note added in proof: Histological, immunohistochemical,and electrophoretical findings on atherectomy speci-mens from primary stenosing lesions are now in press:Dartsch PC, Bauriedel G, Schinko I, Weiss HD, Hofling B,Betz E: Cell constitution and characteristics of humanatherosclerotic plaques selectively removed by percuta-neous atherectomy. Atherosclerosis (in press).

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Index Terms: arterial occlusive disease • arteriosclerosis • cell culture • percutaneous atherectomy •smooth muscle cells



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P C Dartsch, R Voisard, G Bauriedel, B Höfling and E Betzhuman primary stenosing and restenosing lesions.

Growth characteristics and cytoskeletal organization of cultured smooth muscle cells from

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