A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis

ELSEVIER

International Journal of Cardiology 51 (1995) 15-28

A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis Rainer Voisard* a, Uh-ich Seitzer a, Regine Baur a, Peter C. Dartschb, Hans Osterhuesa, Martin Hbhera, Vinzenz Hornbach” aDepartment

of

Cardiology,

bInstitute

Angiology,

of Physiology

Nephrology, and Pneumology, University of Urn, Robert-Kochstra$e 8, D-89081 Federal Republic of Germany I, University of Twinge% Grnclinstr. 5. D-72076, Federal Republic of Germany

Urn,

Received28 February 1995;accepted10 May 1995

Abstract Background. Recentadvancesin the understandingof the biology of restenosis indicatethat it is predominantlycaused by a multifactorial stimulationof smoothmusclecell proliferation. The aim of this study wasto investigatethe in vitro effect of five potential antiproliferative agentson smooth musclecellsfrom human atheroscleroticfemoral arteries.Methods and results. Primary stenosingplaquematerial of 24 patients(aged63 =t 14years)and restenosing plaque material of 7 patients (aged 65 f 9 years) was selectivelyextracted from femoral arteriesby the Simpson atherectomydevice.Cellswereisolatedby enzymaticdisaggregationand identified assmoothmusclecellsby positive reaction with smoothmusclecu-actin.Dalteparin sodium(0.001-100anti-Xa units/ml), cyclosporineA (0.005-500 &ml), colchicine(0.00004-4pgml), etoposide(0.002-200&ml), and doxorubicin (0.0005-50pglml) wereaddedto the cultures.Six days after seeding,cellswere trypsinized and cell numberwasmeasuredby a cell counter. All five agentstestedexhibited a significantinhibition of smoothmusclecell proliferation (P c 0.001).After an incubation time of 48 h, the cytoskeletalcomponents,a-actin, vimentin, and microtubuleswereinvestigated.At peak concentrations, all five testedagentsexceptdalteparin sodiumcausedseveredamageto the cytoskeleton.Conclusions. All five potential antiproliferative agentsexhibited a significantinhibition of smoothmusclecell proliferation. The developmentof newintravasculardelivery systems may opentheway for localantiproliferative treatmentstrategiesin interventional cardiology. Keywords:

Restenosis;Smoothmusclecells;Prescreening;Antiproliferative therapy

* Corresponding author.Tel.:0731/502 4441;,Fax: 0731/502 4442.

0167-5273/951’.W9.50 0 1995ElsevierScience IrelandLtd. All rightsreserved SSDI

0167-5273(95)02377-Z

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1. Introduction

The major limiting factor of percutaneous transluminal coronary angioplasty (PTCA) is the occurrence of restenosis in 30% to 50% of cases within the first 6 months [1,2], adding greatly to the morbidity and cost of the procedure [3]. Despite much investigative work, little progress has been made in this field since the introduction of PTCA in 1977 [4]. The process of restenosis is complex and not fully understood. After vascular injury from balloon angioplasty, the process of restenosis is initiated with the release of thrombogenic, vasoactive, and mitogenic factors [5]. Endothelial and deep-vessel injury leads to platelet aggregation, thrombus formation, inflammation, and activation of smooth muscle cells (SMCs). These events induce the production and release of growth factors and cytokines, which in turn may promote their own synthesis and release from target cells. Thus, a self-perpetuating process is initiated 161. This response to injury resembles atherosclerosis, and restenosis has been considered an accelerated version of this process [7]. Experimental in vivo [8,9] and in vitro studies [lO,l l] suggest a direct relationship between intimal hyperplasia and vessel wall injury at intervention. Human cell culture studies comparing primary stenosing and restenosing plaque material have demonstrated that SMCs from restenosing atherosclerotic tissue show a considerably enhanced proliferative activity in comparison to SMCs from primary stenosing tissue [12,13]. Most of the new non-balloon devices tend to add further injury to the tissue [ 141; therefore, it is not surprising that new mechanical, thermal, and laser devices were not able to reduce the rate of restenosis [ 15,161. Despite numerous randomized trials on systemic pharmacological interventions, the frequency of restenosis has not diminished since the inception of PTCA 15 years ago [ 171. Since systemic administration of antiproliferative agents is limited due to serious side effects, a considerable number of experimental and clinical investigations will be started in the near future to find effective drugs for local drug delivery systems. Cell culture experiments with SMCs from human

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atherosclerotic plaque material allow rapid screening of various drugs and can offer valuable data for effective drug concentrations [ 181. The present study was undertaken to investigate the in vitro effect of dalteparin sodium, cyclosporin A, colchicine, etoposide, and doxorubicin on SMCs from human peripheral plaque material. 2. Methods 2.1. Extraction of human plaque material Plaque tissue from advanced primary stenosing and fresh restenosing lesions of superficial femoral arteries was extracted with a Simpson atherectomy device. A total of 170 specimens from primary stenosing lesions of 24 patients (ages: 63 f 14 years) and 52 specimens from restenosing lesions of 7 patients (ages: 65 l 9 years) were obtained at the Department of Internal Medicine I, University of Munich, FRG, and the Department of Internal Medicine II, University of Ulm, FRG. The success of the Simpson device was controlled by angiographic parameters: Prior to percutaneous atherectomy the average stenosis rate was 93% f 8% and decreased to 21% f 12% following the intervention. 2.2. Cell isolation, cultivation and ident$cation Immediately after extraction, plaque specimens were transferred to sterile glass flasks containing HEPES-buffered culture medium (15 mM). The flasks were transported within 3 to 6 h to the cell culture laboratory. Cells were isolated by a collagenase-elastase enzyme mixture and identified as SMCs by positive reaction with smooth muscle ar-actin, as described elsewhere [12]. All cell culture experiments were carried out with the permission of the Ethical Committee of the University of Ulm and Tiibingen and informed consent was obtained from all patients before the procedure. 2.3. Agents The following drugs were used for cell culture studies: (1) dalteparin sodium, molecular weight approximately 5 kDa, specific activity 167 anti-Xa units/mg (Pfrimmer Kabi, Erlangen, FRG),at concentrations ranging from 0.00 1- 100 anti-Xa’unitsl ml (which equals 0.006-600 &ml); (2) cyclospo-

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rine A (Sandoz, Nuemberg, FRG) at concentrations ranging from 0.005-500 &nl; (3) colchicine (Sigma, Deisenhofen, FRG) at concentrations ranging from 0.00004-4 pg/ml; (4) etoposide (Bristol, Munich, FRG) at concentrations ranging from 0.002-200 j@nl; (5) doxorubicin (Farmitalia, Freiburg, FRG) at concentrations ranging from 0.0005-50 &nl. Drugs were dissolved in distilled water or 99.8% ethanol and added to the culture medium in appropriate concentrations. Control dishes were cultured with an equivalent amount of solvent without the drug. Table 1 Presentation

of clinical

Patient

Age 64

Primary 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

stenosing 73 63 45 70 64 48 73 64 61 80 48 63 68 75 64 38 78 89 60 58 30 50 80 69

Restenotic 2.5 26 27 28 29 30 31

data from

with

advanced

primary

51 (1995)

F, female;

stenoses and fresh restenoses

Sex

Stenosis (%) Before/after

Number of specimens

Weight (w)

F M M F M F M F M M M M F F M F F M M M M M F M

95115 loo/15 100/30 100/30 90125 lOO/lO 100/30 loo/30 loo/15 80/30 loo/50 loo/30 90/20 loo/30 95115 loo/25 95115 loo/30 70120 99121 so/40 90/30 so/20 go/20

5 14 7 4 8 3 21 22 7 14 5 11 8 9 2 2 3 3 10 2 3 2 2 3

52 184 81 57 94 21 167 347 51 124 43 98 nd 88 102 14 8 33 36 21 18 25 16 26

M F M M F M F

no data 90/o 95110 80/35 95/o 90110 90/o

M, male.

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17

2.4. Cytoskeletal components SMCs were seeded at a density of 1000 to 5000 cells/cm2 on round cover slips and incubated in 6well plates for two days for complete cell attachment and spreading. Thereafter, cells were treated for 48 h with dalteparin sodium (100 III/ml), cyclosporine A (500 &nl), colchicine (4 pg/ml), etoposide (200 @g/ml), and doxorubicin (50 pg/ml). After fixation in methanol for 6 min at -2O”C, indirect immunofluorescence was carried out as previously described [ 191. The following primary antibodies were used at a concentration of 10

jesions

lesions 62 73 52 59 73 59 80

nd, not done;

patients

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nd 95 79 23 58 30 130

of plaque

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&nl: (1) monoclonal anti-a-smooth muscle actin (Progen Biotechnik, Heidelberg, FRG), (2) monoclonal anti-vimentin (Camon, Wiesbaden, FRG), (3) monoclonal anti-desmin (Dianova, Hamburg, FRG), (4) monoclonal anti-a-tubulin (Amersham Buchler, Braunschweig, FRG). Tetrareodaminylisothiocyanate (TRITC)- and fluorescein isothiocyanate (FITC)-labeled second antibodies (goat anti-mouse IgG) were purchased from Miles Scientific, Munich, FRG, and Dianova, Hamburg, FRG. Cells were mounted in Mowiol 4-88 [19] and examined with a Nikon Optiphot microscope equipped for epifluorescence with appropriate filter sets. Per agent and per antibody cytoskeletal components of 100 cells were investigated according to the criteria ‘cytoskeleton intact’ or ‘cytoskeleton changed’. 2.5. Measurement

of cell size and cell proliferation

For the examination of cell size and cell proliferation after drug-treatment, SMCs were seeded into 6-well plates (Costar 3406, Technomara, Fernwald, FRG) at a density of 2000 to 3000 cells/cm2. One day after seeding, the culture medium was exchanged and drugs were added at concentrations as described above. At each medium exchange, drugs were renewed as well. Six days after seeding, cells were washed twice with phosphate buffered saline (PBS) and detached by trypsinethylenediamine tetraacetic acid (EDTA) treatment. Cell number and cell size of the single-cell suspension was measured in a cell analyzer system (CASY l/2.04, Scharfe System, Reutlingen, FRG). 2.6. Statistical

evaluation

Results are expressed as mean f S.D. Statistical significance of differences between controls and drug-treated cells was determined by Student’s t-test. Differences were considered signifcant at a value of P < 0.05 [20].

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Fig. 1. Cultured smooth muscle sclerotic tissue exhibit the typical Bar = 400 am.

cells from human ‘Hill and Valley’

atheropattern.

primary stenosing lesions (n = 170) of 24 patients (ages: 65 f 14 years) and 52 specimens from restenosing lesions were obtained at the Department of Internal Medicine I, University of Munich, FRG, and at the Department of Internal Medicine II, University of Ulm, FRG (Table 1). In all cases, only approximately 30 000 cells/100 mg of plaque tissue were isolated by enzymatic disaggregation. Cultured SMCs exhibited their typical ‘hill and valley’ growth pattern (Fig. 1). SMCs from primary stenosing lesions became senescent after reaching passage 2 (according 4 to 5 population doublings). In contrast, SMCs from restenosing lesions could be subcultured several times and exhibited an extraordinarily high proliferation rate. After 5 passages, the first signs of the beginning senescence were observed, such as a decreased capacity for further cell doublings and an abundant amount of cell debris. By passages 8 to 10, SMCs from restenosing plaque material became quiescent. For comparable proliferation studies with different agents, only SMCs from restenosing plaque material in passages 2 to 4 could be used.

3. Results 3.2. Cell proliferation 3.1. Cell culture

For the selective extraction of primary and restenosing plaque tissue of superficial femoral arteries, a Simpson atherectomy device was used as described previously [2 1,221. Specimens from

after drug application

After an incubation period of 5 days, a significant inhibition of SMC proliferation was caused by all five agents tested. The concentrationresponse curve of low-molecular-weight heparin (LMWH, Fig. 2A) showed no inhibition of SMC

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19

120

*

100

C r; "E

00

z =

60

t

c

0.001

Concentration

0.01

1

0.10

10

of low-molecular-weight

f 3 3

40

i

20

*

0

100

c

heparin

0,0005

Concentration

0,005

0.05

0.5

of cyclosporine

5

50

A in Ipg/mU

in [ IU 120

120

C *

I3

100

.c 2t

so

z =

60

** * u--l

Y f P 9

40

p

20

0 c

0.000040.00040.004

Concentration

0.04

of colchicine 120

0.4

E =

60

G f 'Z 3

40

fz

20

4

0 c

in Ipg/mll

0,002

Concentration

0.02

02

of etoposide

2

20

200

in [pg/mll

I

I

E

c 0.00050.0050.05 0.5 5 Concentration

of doxorubicin

50

in [pg/mll

Fig. 2. Effect of dalteparin sodium (A), cyclosporine A (B), colchicine (C), etoposide (D), and doxorubicin of smooth muscle cells from human atherosclerotic tissue. The untreated control after 5 days incubation mean values f S.D. of pooled cells of different donors. *P < 0.001; Student’s t-test.

(E) on the proliferation is 100%. Data. represents

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proliferation at concentrations ranging from 0.001 to 0.1 IU/ml. However LMWH-concentrations of 1, 10, and 100 III/ml caused a significant, dosedependent inhibition of SMC proliferation (P < 0.001; Student’s t-test), reaching 40% at a concentration of 100 IU/ml. At low concentrations of cyclosporine A (0.0005 to 0.5 &ml), there was a small but insignificant inhibition of SMC proliferation (Fig. 2B), whereas cyclosporine A at a concentration of 5 &ml inhibited SMC proliferation by more than 40% (P c 0.001; Student’s t-test). At peak concentrations of cyclosporine A (50 pg/ml), SMC proliferation was inhibited by more than 90% in comparison with control dishes (P < 0.001; Student’s t-test). Colchicine (Fig. 2C), at a concentration of 0.00004 &ml, had no effect on cell proliferation. However, a small, but insignificant inhibition of SMC proliferation was observed at a concentration of colchicine of 0.0004 and 0.004 pg/ml. A clear inhibition of cell proliferation by more than 40% (P < 0.001; Student’s t-test) was caused by colchicine at a concentration of 0.04 pg./ml. The further increases of concentration of colchicine to 0.4 and 4 &ml did not enhance the inhibitory effect. Already the lowest tested concentration of etoposide (0.002 &ml) caused a signiticant inhibition of SMC proliferation (P < 0.001; Student’s r-test), and etoposide (Fig. 2D) at a concentration of 0.02 pg/ ml inhibited SMC proliferation by 50% (P < 0.001; Student’s t-test). Although further increases of etoposide concentrations (0.2,2, and 20 pg/ml) did not enhance the inhibitory effect, the maximum concentration of 200 &ml etoposide inhibited proliferation of SMCs by almost 80% (P < 0.001; Student’s t-test). Doxorubicin (Fig. 2E) at the lowest tested concentration (0.0005 yg/ml) had no effect on the proliferative activity of smooth muscle cells. However, at a concentration of 0.005 &nl, there was already an inhibition of SMC proliferation by almost 50% (P < 0.001; Student’s r-test); a further increase to a concentration of 0.05 pgml did not cause more growth inhibition. However, a further increase of drug concentration resulted in a dosedependent decrease in proliferative activity. At a concentration of 50 &ml doxorubicin, proliferation of SMCs was inhibited by more than 90% (P < 0.001; Student’s t-test).

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3.3. Cell morphology after drug application

With every concentration of dalteparin sodium, cell morphology was not altered at light microscopic levels. Whereas partial damage of cell morphology was caused by low concentrations of etoposide (0.002 &nl), mean concentrations of colchicine, etoposide, and doxorubicin severely affected cell morphology. Cells became rounded and remained slightly attached or underwent complete lysis. Peak concentrations of cyclosporine A had the same drastic effect on cell morphology. With increasing time of cultivation, the number of viable and still adherent cells was reduced in a dose-dependent manner. Fully spread inter-phase cells seemed to be less sensitive than mitotic cells that were rounded and anchored with thin filaments. 3.4. Cytoskeletal alterations after drug application

Cytoskeletal features of SMCs were characterized by indirect immunofluorescence after high-dose treatment with antiproliferative agents (Table 2). The cytoskeleton of SMCs is made up of actin filaments with a diameter of about 6 nm, intermediate tilaents with a diameter of about 7-l 1 nm, and microtubules with a diameter of about 20 nm (Fig. 3A,B,C). Actin filaments in control dishes without agents (Fig. 3A) appeared as uniform threads that were arranged longitudinally through the cytoplasma. Actin filaments (Table 2) were almost completely destroyed by peak concentrations of colchicine (4 pg/ ml, Fig. 3D), cyclosporine A (50 &ml), etoposide (200 hg/ml), and doxorubicin (50 Table 2 Effect of potential antiproliferative drugs on the cytoskeleton Filaments LMW-heparin (loo IU/rtll) Colchicine (4 &ml) Cyclosporine A (50 Pg/ml) Etoposide

@JoPglml)

Doxorubicin (50 cd-4

or-Actin

Vimentin

cz-Tubulin

0%

0%

0%

91% 98%

99% 96%

100% 100%

89%

4%

15%

90%

89%

89%

Change of cytoskeletal cell structure in percent after incubation for 48 h.

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~g/rnl, Fig. 3G), whereas peak concentrations of dalteparin sodium (100 W/ml) had no visible effect. Intermediate filaments consist of 5 different groups and range in size between actin filaments and microtubules. In control dishes without drugs, vimentin (Fig. 3B) was observed to form a typical basket around the nucleus and to extend in curving arrays to the cell’s periphery, as described elsewhere [ 121. After an incubation period of 48 h, vimentin structures (Table 2) were severely damaged in more than 90% of cells by colchicine (4 pg/ml, Fig. 3E) and cyclosporine A (50 kg/ml), and in almost 90% by doxorubicin (50 pg/ml, Fig. 3H). Surprisingly, etoposide at a concentration of 200 pg/rnl revealed only some effect on vimentin structures in 4%, whereas dalteparin sodium (100 IU/ml) caused no effect. Desmin, another intermediate filament, was not identified in SMC cultures. Microtubules in control dishes (Fig. 3C) radiated from the perinuclear microtubule-organizing centers (MTOC) out into the cell’s periphery in fine lacelike threads [ 121 and were typically seen in greatest density around the nucleus. Damage to the microtubule system (Table 2) was seen in 100% of the investigated SMC after a 48 h incubation time with colchicine (4 pg/m.l, Fig. 3F) and doxorubicin (50 &ml) and in 89O/o after treatment with doxorubicin (50 pg/rnl, Fig. 31). Surprisingly, etoposide at a concentration of 200 &ml damaged microtubules in only 15%, whereas no visible effect was seen after incubation with dalteparin sodium at a concentration of 100 IU/ml. 4. Discussion

Human cell culture studies have demonstrated that SMCs from restenosing atherosclerotic tissue of peripheral and coronary arteries show a considerably enhanced proliferative activity in comparison with SMCs from primary stenosing tissue [12,13,23]. In the literature, it is reported that the maximum of smooth muscle cell proliferation in vivo takes place during the first week after ballooning [24]. Therefore, we investigated the in vitro effect of potential anti-proliferative agents 6 days after seeding. The major finding of this study is that all 5 drugs tested caused a significant inhibition of SMC proliferation.

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4.1. Low-molecular-weight heparin Heparin is a glycosoaminoglycan composed of repeating glucosamine and glucuronic acid sugar residues. Heparin and heparin-like molecules produced by endothelial cells inhibit SMC growth in vitro [25] and SMCs themselves synthesize heparan sulfate, which shows growth inhibitory effects [26]. In a rat carotid artery injury model, Clowes and Karnovsky [27] first demonstrated the inhibitory effect of heparin on myointimal thickening. In vitro studies by this group have demonstrated a dose-response relation [25] that was also enhanced by pretreatment with heparin for 48 h [28]. Another potentially important effect of heparin is the inhibition of migration of SMCs from the media to the intima [29]. Many mechanisms have been described for heparin growth inhibition. Recent studies [30] have shown that SMCs bind and internalize heparin, consistent with receptor-mediated endocytosis. Of interest, growth-arrested SMCs bound significantly more heparin than did exponentially growing cells and were more sensitive to the growth inhibitory effects of heparin [25,30]. These data suggest that maximal effectiveness of clinical heparin requires administration before SMC proliferation. Controversy exists regarding the phase of the cell cycle that is inhibited by heparin: Reilly et al. [31] presented evidence for prevention of entry into the S phase without effect on early proto-oncogene expression (c-fos and c-myc), whereas Pukac et al. [32] showed inhibition of cfos and c-myc expression, events characteristic of progression from Go to G1. Bennet et al. [33] recently demonstrated by a variety of different techniques that constitutive expression of c-myc, even at low levels seen in normal proliferating vascular SMCs, blocks growth arrest in response to treatment with heparin. The currently available clinical data on heparin and restenosis show conflicting results. Hirshfeld et al. [34] suggested that there may be a reduced incidence of restenosis after PTCA in patients with prolonged heparin treatment after intervention, whereas in a study of Ellis et al. [35], no reduction of restenoses was found. Low molecular weight fractions of heparin (2000 to 5000 Da) retain the anti-factor Xa activ-

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Fig. 3. Cytoskeletal architecture of smooth muscle cells before (A,B,C) and after treatment with colchicine in a concentration of 4 kg/ml (D,E,F) and doxorubicin in a concentration of 50 pg/ml (G,H,I). Depicted is the effect of colchicine on o-actin (D), vimentin (E), and microtubules (F) and the effect of doxorubicin on o-actin (G). vimentin (H), and microtubules (I). Bar = 30 pm.

ity, which some investigators believe to account for the antithrombotic effect of heparin [36]. Although these ‘heparinoids’ lack anticoagulant properties, they still inhibit SMC growth in culture [37]. LMWH-treatment is reported to reduce the incidence of restenosis after balloon angioplasty in rabbits by histological and angiographic criteria [38]. In a rabbit model, it was also demonstrated that LMWH treatment results in a significant inhibition of SMC proliferation 3 and 7 days after angioplasty [39], suggesting an early LMWH treatment after angioplasty to prevent restenosis. LMWH has been investigated in cell cultures with SMCs from the media of undiseased human aortas [40]. The findings of this study are in accordance with our data, demonstrating a significant

inhibition of SMC proliferation (20%) at a concentration of 3.9 &ml (which equals 0.62 anti-Xa IU/ml) and a 40% inhibition at a concentration of 390 &ml (which equals 62 anti-Xa III/ml). Chan et al. recently reported [41] that high concentrations of heparin (100 &ml) inhibit the proliferation of SMCs from human restenotic lesions by only 8%. Although the results cannot be easily compared, since we investigated LMWH and not heparin, the reduced heparin sensitivity of some SMCs from human restenotic arteries is very interesting. The different results may be explained by findings of San Antonio et al. [42], who described the isolation of heparin insensitive SMCs in vitro and suggested that after vascular injury, the loss of SMC growth control by heparin and heparin

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Fig. 3 (continued)

sulfates may trigger the increased SMC proliferation in restenotic lesions. 4.2. Cyclosporin

A

The rationale for the use of antiinflammatory agents is that they inhibit cell accumulation and

activation at the site of vascular injury 1431. Steroid agents inhibit the proliferation of smooth muscle cells from human restenotic lesions [44] and the combination of corticosteroids and heparin inhibits smooth muscle cell proliferation in animals [45]. Human atherosclerotic plaques contain

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a high proportion of T lymphocytes, which may modulate the vascular response after injury [46]. In response to balloon catheter-induced arterial injury in rat, SMCs express class II antigens at the same time as T cells are infiltrating the experimental lesions [47]. This indicates that T cells can control gene expression in SMCs and suggests that such a mechanism is operating in the atherosclerotic plaque. Consequently, it was postulated that suppression of T cell activation might modulate the arterial response to injury. Cyclosporin A inhibits the interleukin 2-mediated autocrine stimulation of T cell growth during activation of the T cell, as well as the secretion of other T cell lymphokines [48,49]. In vivo and in vitro investigations with cyclosporine A have been contradicting. Jonasson et al. [50] have reported a decrease in intimal proliferative thickness in carotid artery de-endothelialization in rats with high doses of cyclosporin (1.4 ~.g/ml), but did not find any direct effects of cyclosporin A at a concentration of 2 &ml on proliferation of SMCs in vitro. Although Jonasson et al. [50] isolated SMCs from the undiseased media of rats, whereas we used SMCs from human atherosclerotic lesions, the results are not in contrast to our results. We found that cyclosporin A at a concentration of 0.5 &ml did not cause a significant inhibition of SMC proliferation, whereas cyclosporin A at a concentration of 5 &ml revealed a significant inhibition to almost 50%. The different results may be explained by different drug concentrations: Jonasson et al. [50] used cyclosporin A at a concentration of only 2 &ml, whereas we tested concentrations twice and ten times higher. In consequence, it seems reasonable to suggest that cyclosporin A inhibits the arterial response to injury by blocking the T cell-mediated control of vascular proliferation via the immune system already at a concentration of 1.4 &ml [50], whereas a direct inhibition of SMC proliferation can be seen at a concentration of 5 &ml. 4.3. Colchicine Colchicine is an alkaloid derived from the plant Colchicium autumnale. It is an antimitqgenic agent that binds to tubulin, disrupting spindle formation and resulting in the metaphase arrest of cell

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division. Colchicine has been shown to inhibit chemotaxis [51,52], collagen formation [53], muscle cell proliferation and platelet aggregation [54,55]. In experimental animal models, this agent has prevented or reduced the formation of atherosclerotic plaques [56,57]. Colchicine has also been effective in preventing myointimal proliferation after balloon arterial injury of the iliac artery in an atherosclerotic rabbit model [58]. However, this effect was only apparent with the high dose of 0.2 mglkg per day (corresponding to about 2 pg/m.l) and not with the low dose of 0.02 mg/kg per day (corresponding to about 0.2 j&ml). High dose colchicine has been used as an antineoplastic agent for conditions such as leukemia [59], but was poorly tolerated because of serious adverse effects. Even in a large clinical trial with 145 patients [60], the oral application of the low dose of 1.2 mg colchicine caused severe gastrointestinal adverse effects, but had no beneficial effect for preventing restenosis after coronary angioplasty. Since in the standard 60 kg human, oral treatment with 1.2 mg colchicine daily corresponds with a plasma level of about 0.2 &ml, with regard to the findings of Currier et al. [58], these results had to be expected. However, in our investigation, colchicine already inhibited SMC proliferation by more than 60% at a concentration of 0.04 pg/ml. The reason for the high sensitivity of cultured smooth muscle cells remains unclear, but might be caused by a direct effect of colchicine on the cultured cells, which cannot occur in the same manner with in vivo conditions.

4.4. Cytotoxic agents The use of more potent antimitotic agents for the prevention of restenosis has not been well studied, predominantly because of the potential hazards of such toxic therapy and the relatively benign clinical consequences of restenosis. The hyperplastic SMCs responsible for the restenotic process are of mesenchymal cell origin [61]. The agents generally used for tumors arising from mesenchymal cells include methotrexate, vincristine, cyclophosphamide, and anthracycline antibiotics. Accordingly, the antineoplastic agents

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investigated thus far in animals have been from this group of drugs. The systemic use of a combination therapy with vincristine and actinomycin D has been evaluated in a rabbit aortic model [62]. Short-term therapy (3 days after endothelial denudation) resulted in less smooth muscle cell hyperplasia in the treated animals [62]. The intermediate and long-term effects of this therapy were not observed in this study. The effectiveness of local methotrexate therapy on intimal proliferation after balloon arterial injury was evaluated by Muller et al. [63]. The antimetabolite methotrexate was administered locally through a Wolinsky coronary infusion balloon catheter 1641. In this model, local infusion of methotrexate did not abolish or even attenuate intimal proliferation. The use of systemic antineoplastic agents for restenosis was also addressed by Murphy et al. [65]. Their restenosis model was created by oversized stents placed in the porcine coronary artery. In this study, the use of oral or intramuscular methotrexate or azathioprine did not inhibit porcine intimal proliferation and restenosis. In an experimental study with cholesterol-fed rabbits, it has been demonstrated by Llera-Moya et al. [66] that subcutaneouslyadministered etoposide treatment suppresses atherosclerotic plaque development. These effects were independent of the extent of the diet-induced hyperlipemia or an effect of etoposide on blood cell count and seem to be related to an inhibition of intimal cell proliferation 1661. In our investigations, we have found that the proliferation of SMCs derived from human plaque material is inhibited by more than 50% by a concentration of 0.02 &ml, which is much lower than the concentration used by Llera-Moya et al. [66]. It should be tested in further in vivo investigations whether a low-dose etoposide treatment will suppress atherosclerotic plaque development as well. In vitro studies with SMCs derived from primary stenosing human coronary plaque material have already shown a strong anti-proliferative effect of cytarabine, doxorubicin, and vincristine [67]. Doxorubicin (0.5 &ml) inhibited SMC proliferation of primary stenosing plaque material by 50%, whereas our data demonstrate an inhibition of more than 60%. The difference might be caused

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by the fact that we used cells isolated from restenosing plaque material, which possess an increased proliferative activity 1121 and therefore a higher sensitivity to antineoplastic agents. 4.5. Cytoskeletal features In our experimental conditions, more than 80% of all cultured cells stained positive with antibodies against smooth muscle a-actin. In the literature, reports on the amount of smooth cells in primary cultures are contradictory and vary from l5%-30% [68] to over 90% [12]. The striking difference to the results of Pickering et al. [68] remains unclear, but might be partially explained by the fact that Pickering et al. used the explant technique whereas we used enzymatic disaggregation, and thus different cell types might have been selected. In agreement with reports of a decrease of desmin during human atherosclerotic plaque development [ 12,691, SMCs stained positive with antibodies against vimentin but negative with antibodies against desmin. It has been reported that vascular damage at all stages of atherosclerosis may cause antibody production against unmasked SMC cytoskeletal components, especially against desmin [69]. It is now generally accepted that cytoskeletal markers are reliable tools for a better understanding of subcellular drug effects. LMWH had no visible effect on the microtubule system, although SMC proliferation was inhibited to 40% at peak concentrations. This seems to indicate that the physiologic functions of microtubules are much earlier affected than might be expected from the visible damage. The drastic effects of peak concentrations of colchicine, cyclosporine A, and doxorubicin on the microtubules correspond with the strong antiproliferative effect of these agents. 4.6. Limitations of the study Considerable caution should be exercised in the extrapolation of positive results in experimental in vitro studies to the likelihood of a drug’s efficacy in humans. A very strbng inhibition of SMC proliferation in vitro might turn out to cause toxic effects with a subsequent rupture of the vessel wall in vivo. Furthermore, the complex interactions between SMCs, endothelial cells, and macro-

26

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phages cannot be observed in a monoculture model. Transfilter co-culture [70,71,72] and organculture systems [lO,l l] offer a striking similarity with the cellular responses of the in vivo situation. Therefore, these systems seem to be suitable to obtain further insight into basic problems of postangioplasty cell proliferation and the development of preventive strategies [73]. 4.7. Implications of the study Peak concentrations of all five agents tested caused a clear inhibition of SMC proliferation in vitro. Because of serious side effects, a systemic application of these concentrations for the prevention of postangioplasty cell proliferation cannot be recommended. However, new developments in catheter design, molecular biology, and polymer chemistry have made it possible to deliver pharmaceutical agents and genetic material directly into the wall to modulate the response to injury [74,75]. Several local drug delivery catheters of various designs in addition to biodegradable and coated stents are currently being evaluated as devices to facilitate local delivery of agents into the arterial wall. Recently, it has been reported that the local administration of heparin [76], doxorubicin [77], and colchicine [78] with the Wolinsky infusion catheter [64] did not reduce the extent of intima thickening after angioplasty in the rabbit model. The failure might have been caused by an additional injury of the artery wall by the porous catheter system [63] and/or by insufficient drug concentrations. Our data may offer valuable information for a sufticient dose-finding in further in vitro and in vivo investigations in the fascinating field of site-specific therapy for the prevention of restenosis. Acknowledgements The authors are indebted to Berthold Hofling, MD and Gerhard Bauriedel, MD, University of Munich, .,FRG, and to Hans Osterhues, MD, University of Ulm, FRG, for the extraction of plaque material from peripheral arteries. This study was supported by grant 26 from the Ministerium fur Wissenschaft und Kunst des Landes BadenWuerttemberg, Federal Republic of Germany.

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