Experimental Assessment of Effects of Antiproliferative Drugs Used in

Drug-Eluting Stents on Endothelial Cells

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Abstract

Background: Late and very late stent thrombosis after drug-eluting stent implantation is a

major concern. The present study evaluated difference in the effects of sirolimus, paclitaxel

and zotarolimus on endothelial cells.

Methods: Mouse endothelial cells were seeded in a 6-well plate. Cells were cultured with an

antiproliferative drug at the expected concentrations for each well for 24 hours before making

3 scratch lines with a pipette tip. After a 4.5 hour incubation period, 3 reference scratch lines,

vertically across the original scratch lines, were made in the same way. The experiment was

repeated at least 6 times (6 plates). Measurements were performed at 9 crossings of each well.

Wound healing ratio was calculated as 1 - (distance of the first scratch/distance of the second

scratch). % cell migration was calculated as (wound healing ratio at an expected drug

concentration/wound healing ratio with no drug) × 100. Average % cell migration at 54

crossings of 6 plates was calculated.

Results: Paclitaxel inhibited cell migration in a concentration-dependent manner. On the other

hand, concentration-dependent inhibition was not observed for sirolimus or zotarolimus.

Sirolimus showed a stronger inhibitory effect on migration of endothelial cells compared to

zotarolimus.

Conclusions: The difference in the effect of antiproliferative drugs of drug-eluting stents on

endothelial cells may be associated with relatively faster re-endothelialization of

zotarolimus-eluting stent compared to the 1st generation DES.

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1. Introduction

Drug-eluting stent (DES) has dramatically reduced in-stent restenosis. However, late

and very late stent thrombosis after DES implantation has emerged as a major concern. The

antiproliferative drugs used in DES not only inhibit intimal hyperplasia but also delay

re-endothelialization. Recent trials have shown a lower stent thrombosis rate of the 2nd

generation DES compared to the 1st generation DES [1]. Thus the effect of the

antiproliferative drugs of the 1st and 2nd generation DES on re-endothelialization may be

different. The present experimental study evaluated difference in the effect of the

antiproliferative drugs of the 1st and 2nd generation DES on endothelial cells.

2. Methods

2.1. Chemicals

Chemicals were purchased from the following commercial sources: paclitaxel (Wako

Chemicals, Japan), zotarolimus (Toronto Research Chemicals, Canada), and rapamycin

(Sigma, USA). Ethanol was used as solvent for paclitaxel and rapamycin, and DMSO for

zotarolimus.

2.2. Cell culture

Mouse

BRC through the National Bio-Resource Project of the MEXT, Japan. Cells were cultured at

37 °C in a 5% CO2, humidified atmosphere. Cells were grown in Dulbecco’s Modified

Eagle’s Medium (High glucose, Sigma, USA) with 10% fetal bovine serum (HyClone, USA),

100 units/ml penicillin, and 100 μg/ml streptomycin from Nacalai Tesque (Kyoto, Japan).

2.3. Scratch assay

We assessed the effects of antiproliferative drugs of DES on cultured endothelial

cells by scratch wound assay. Cells were seeded at the density of 0.7-1.5 105 cells/well in a

6-well plate. Following the pre-culture period for 24 hour (Figure 1), the volume of culture

medium in each well was adjusted to 2 ml to start the incubation of cells with a drug at the

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expected concentrations for each well in the plate. The drug concentrations were selected

according to those in previous studies [2,3]. Cells were cultured for another 24 hours before

making 3 scratch lines with a pipette tip (200-μl size), and the closure of the scratch lines was

measured after a 4.5-hour incubation period. Three reference scratch lines, vertically across

the original scratch lines, were made in the same way just before fixing cells with 4%

paraformaldehyde for 10 min at room temperature prior to take images under the microscope

at 40× magnification (Figure 2) [4]. The images acquired for each sample were analyzed

quantitatively by using ImageJ 1.48v (NIH, USA). The experiment was repeated at least 6

times (6 plates).

Cell migration was evaluated by comparing the images between the solvent-treated

control cells and the drug-treated cells. For each image, measurements were performed at 9

crossings of each well (Figure 1). The distances of the first scratch and the second scratch

were measured at 6 points (Figure 2). The mean distances of the first scratch and the second

scratch were calculated. Wound healing ratio was calculated as 1 - (mean distance of the first

scratch/mean distance of the second scratch). % cell migration was calculated as (wound

healing ratio at an expected drug concentration/wound healing ratio with no drug) × 100.

Average % cell migration at 54 crossings of 6 plates was calculated.

2.4. Statistical analysis

Statistical analysis was performed with SAS9.4 (SAS Institute Inc. Cary, NC, USA).

Values are shown as mean ± SD. Continuous variables were compared using Student’s t test

or one-way analysis of variance. Statistical significance was defined as p <0.05

3. Results

Paclitaxel inhibited cell migration in a concentration-dependent manner (Figure 3).

On the other hand, concentration-dependent inhibition was not observed for sirolimus or

zotarolimus. Pharmacokinetics of sirolimus-eluting stent and zotarolimus-eluting stent were

reported [5,6]. However, there is little information about pharmacokinetics of

paclitaxel-eluting stent. Thus we compared the effect of sirolimus and zotarolimus at the

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concentrations that were closest but higher than the maximum concentration after sirolimus-

(0.86 0.21 ng/ml) and zotarolimus-eluting stent implantation (1.80 0.53 ng/ml) [6].

Sirolimus showed a stronger inhibitory effect on migration of endothelial cells compared to

zotarolimus (Figure 4), although concentration of zotarolimus compared to that after stent

implantation was much higher than that of sirolimus.

4. Discussion

The present study evaluated effects of antiproliferative drugs of the 1st and 2nd

generation DES on endothelial cells. Inhibition of endothelial cell migration with paclitaxel

was concentration-dependent. The concentration-dependent inhibition was not observed for

sirolimus or zotarolimus. Sirolimus inhibited cell migration more than zotarolimus.

The reported incidence of late and very late stent thrombosis after the 1st generation

DES implantation ranged between 0.2% and 0.7% [7-12]. A cohort study reported that

definite stent thrombosis continued to occur at the constant rate of 0.6% per year from 30

days to 3 years after the 1st generation DES implantation [11]. Stent thrombosis often results

in myocardial infarction or death. Iakovou et al.[12] reported a case fatality rate of stent

thrombosis at 45%. Recent studies have shown that stent thrombosis is less frequent after

implantation of the 2nd generation DES compared to the 1st generation DES [13]. It may be

owing to better stent material, implantation techniques, and antiplatelet therapy. A human

autopsy study has shown greater strut coverage with less inflammation and fibrin deposition

after implantation of the 2nd generation DES compared to the 1st generation DES [14].

DES usually consists of 3 components: (1) metallic stent, (2) drug as an

antiproliferative agent to inhibit neointimal formation, and (3) polymer as a drug-carrier

vehicle. The cause of stent thrombosis is multifactorial. Patient-related, lesion-related, and

procedure-related factors may be intertwined. A study demonstrated less thrombogenicity of

stent with thinner struts [15]. It has been shown that hypersensitivity reaction to the polymer

of the 1st generation stents is associated with late and very late stent thrombosis [16]. However,

delayed re-endothelialization due to the antiproliferative drugs of DES may be mainly

associated with late and very late stent thrombosis [17].

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Paclitaxel inhibits endothelial cell migration and intimal hyperplasia by stabilizing

microtubules [18,19]. Cytostatic effect of low-dose paclitaxel on human endothelial cells has

been demonstrated [20]. Because paclitaxel is an anticancer agent, therapeutic window may be

narrow, which may be associated with delay in re-endothelialization in a

concentration-dependent manner [21]. Sirolimus [22,23] and zotarolimus [24]

inhibit the mammalian target of rapamycin pathway, which results in the antimigration and

antiproliferative effect. A previous study evaluated the effects of antiproliferative drugs of

DES on endothelial progenitor cells [25]. Sirolimus or paclitaxel inhibited endothelial

progenitor cells migration to a significantly greater degree than zotarolimus. Delayed

re-endothelialization of DES is a mechanism of late and very late stent thrombosis. Stronger

inhibitory effect of antiproliferative drugs on migration of endothelial cells may be associated

with delayed re-endothelialization of DES. Cell migration studies may be useful to find a

better antiproliferative drug in case a new DES is developed.

5. Limitations

The present study used mouse vascular endothelial cells and compared the effect of

drugs on cell migration at each concentration based on known pharmacokinetics. The effects

of antiproliferative drugs might be different between mouse and human endothelial

cells. Steinfeld et al.[26] used endothelial cell isolated from human coronary arteries and

reported paclitaxel inhibited cell migration compared with zotarolimus at 1 . The study

compared the effect of drugs on same concentration. However, the effect of actual

concentrations of each antiproliferative drug at the vascular endothelium adjacent to DES is

unknown. In addition, endothelialization can be affected by endothelial progenitor cells and

other kinds of cells. Therefore, further analysis based on more information of

pharmacokinetics of antiproliferative drugs and using those cells may be needed to assess

speed of endothelialization more precisely.

6. Conclusions

Inhibition of endothelial cell migration with paclitaxel is concentration-dependent.

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On the other hand, it is concentration-independent for zotarolimus or sirolimus. Zotarolimus

has less inhibitory effect compared to sirolimus. It may be associated with relatively faster

re-endothelialization of zotarolimus-eluting stent compared to the 1st generation DES.

Acknowledgments

I am deeply grateful to members of Department of Pharmacology, Chiba University

Graduate School of Medicine whose supports were innumerably valuable throughout the

course of my study.

Conflict of interest

This research received no grant from any funding agency in the public, commercial

or not-for-profit sectors. Yoshio Kobayashi received research grant from Medtronic and

Boston Scientific. The other authors declare that there are no conflicts of interest to disclose.

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References

1. Palmerini T, Biondi-Zoccai G, Della Riva D, Stettler C, Sangiorgi D, D'Ascenzo F, et

al. Stent thrombosis with drug-eluting and bare-metal stents: evidence from a

comprehensive network meta-analysis. Lancet. 2012;379:1393-402.

2. Parry TJ, Brosius R, Thyagarajan R, Carter D, Argentieri D, Falotico R, et al.

Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and

injured arteries. Eur J Pharmacol. 2005;524:19-29.

3. Burke SE, Kuntz RE, Schwartz LB. Zotarolimus (ABT-578) eluting stents. Adv Drug

Deliv Rev. 2006;58:437-46.

4. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive

method for analysis of cell migration in vitro. Nat Protoc. 2007;2:329-33.

5. Vetrovec GW, Rizik D, Williard C, Snead D, Piotrovski V, Kopia G. Sirolimus PK

trial: a pharmacokinetic study of the sirolimus-eluting Bx velocity stent in patients

with de novo coronary lesions. Catheter Cardiovasc Interv. 2006;67:32-7.

6. Otsuka Y, Saito S, Nakamura M, Shuto H, Mitsudo K. Comparison of

pharmacokinetics of the limus-eluting stents in Japanese patients. Catheter Cardiovasc

Interv. 2011;78:1078-85.

7. Kitahara H, Kobayashi Y, Fujimoto Y, Nakamura Y, Nakayama T, Kuroda N, et al.

Late stent thrombosis in patients receiving ticlopidine and aspirin after

sirolimus-eluting stent implantation. Circ J. 2008;72:168-9.

8. Ong ATL, McFadden EP, Regar E, de Jaegere PPT, van Domburg RT, Serruys PW.

Late angiographic stent thrombosis (LAST) events with drug-eluting stents. J Am Coll

Cardiol. 2005;45:2088-92.

9. Kuchulakanti PK, Chu WW, Torguson R, Ohlmann P, Rha SW, Clavijo LC, et al.

Correlates and long-term outcomes of angiographically proven stent thrombosis with

sirolimus- and paclitaxel-eluting stents. Circulation. 2006;113:1108-13.

10. Park D-W, Park S-W, Park K-H, Lee B-K, Kim Y-H, Lee CW, et al. Frequency of and

risk factors for stent thrombosis after drug-eluting stent implantation during long-term

8

follow-up. Am J Cardiol. 2006;98:352-6.

11. Daemen J, Wenaweser P, Tsuchida K, Abrecht L, Vaina S, Morger C, et al. Early and

late coronary stent thrombosis of sirolimus-eluting and paclitaxel-eluting stents in

routine clinical practice: data from a large two-institutional cohort study. Lancet.

2007;369:667-78.

12. Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM, Stankovic G, et al. Incidence,

predictors, and outcome of thrombosis after successful implantation of drug-eluting

stents. JAMA. 2005;293:2126-30.

13. Stone GW, Rizvi A, Sudhir K, Newman W, Applegate RJ, Cannon LA, et al.

Randomized comparison of everolimus- and paclitaxel-eluting stents. 2-year

follow-up from the SPIRIT (Clinical Evaluation of the XIENCE V Everolimus Eluting

Coronary Stent System) IV trial. J Am Coll Cardiol. 2011;58:19-25.

14. Otsuka F, Vorpahl M, Nakano M, Foerst J, Newell JB, Sakakura K, et al. Pathology of

Second-Generation Everolimus-Eluting Stents Versus First-Generation Sirolimus- and

Paclitaxel-Eluting Stents in Humans. Circulation. 2014;129:211-23.

15. Kolandaivelu K, Swaminathan R, Gibson WJ, Kolachalama VB, Nguyen-Ehrenreich

KL, Giddings VL, et al. Stent thrombogenicity early in high-risk interventional

settings is driven by stent design and deployment and protected by polymer-drug

coatings. Circulation. 2011;123:1400-9.

16. Virmani R, Guagliumi G, Farb A, Musumeci G, Grieco N, Motta T, et al. Localized

hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent:

should we be cautious? Circulation. 2004;109:701-5.

17. Joner M, Finn AV, Farb A, Mont EK, Kolodgie FD, Ladich E, et al. Pathology of

drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll

Cardiol. 2006;48:193-202.

18. Liuzzo JP, Ambrose JA, Coppola JT. Sirolimus- and taxol-eluting stents differ towards

intimal hyperplasia and re-endothelialization. J Invasive Cardiol. 2005;17:497-502.

19. Kamath K, Smiyun G, Wilson L, Jordan MA. Mechanisms of inhibition of endothelial

cell migration by taxanes. Cytoskeleton (Hoboken). 2014;71:46-60.

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20. Pasquier E, Carré M, Pourroy B, Camoin L, Rebaï O, Briand C, et al. Antiangiogenic

activity of paclitaxel is associated with its cytostatic effect, mediated by the initiation

but not completion of a mitochondrial apoptotic signaling pathway. Mol Cancer Ther.

2004;3:1301-10.

21. Jabara R, Chronos N, Tondato F, Conway D, Molema W, Park K, et al. Toxic vessel

reaction to an absorbable polymer-based paclitaxel-eluting stent in pig coronary

arteries. J Invasive Cardiol. 2006;18:383-90.

22. Moss SC, Lightell DJ, Jr., Marx SO, Marks AR, Woods TC. Rapamycin regulates

endothelial cell migration through regulation of the cyclin-dependent kinase inhibitor

p27Kip1. J Biol Chem. 2010;285:11991-7.

23. Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle

regulators of proliferation in vascular smooth muscle cells. Circ Res. 1995;76:412-7.

24. Chen YW, Smith ML, Sheets M, Ballaron S, Trevillyan JM, Burke SE, et al.

Zotarolimus, a novel sirolimus analogue with potent anti-proliferative activity on

coronary smooth muscle cells and reduced potential for systemic immunosuppression.

J Cardiovasc Pharmacol. 2007;49:228-35.

25. Banerjee S, Xu H, Fuh E, Nguyen KT, Garcia JA, Brilakis ES, et al. Endothelial

progenitor cell response to antiproliferative drug exposure. Atherosclerosis.

2012;225:91-8.

26. Steinfeld DS, Liu AP, Hsu SH, Chan YF, Stankus JJ, Pacetti SD, et al. Comparative

assessment of transient exposure of paclitaxel or zotarolimus on in vitro vascular cell

death, proliferation, migration, and proinflammatory biomarker expression. J

Cardiovasc Pharmacol. 2012;60:179-86.

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Figure legends

Fig. 1 Experimental flow chart.

Fig. 2 Measurements of cell migration. The distances of the first scratch line (W1-6) and

the second scratch line (H1-6) are measured at 6 points. The mean distances of the first

scratch and the second scratch are calculated. Wound healing ratio was calculated as 1 - (mean

distance of the first scratch/mean distance of the second scratch).

Fig. 3 The effect of antiproliferative drugs on endothelial cell migration. Paclitaxel inhibits

cell migration in a concentration-dependent manner. On the other hand,

concentration-dependent inhibition is not observed for sirolimus or zotarolimus.

Fig. 4 The effect of sirolimus and zotarolimus on endothelial cell migration at the each

concentration based on pharmacokinetics after stent implantation. Sirolimus shows a stronger

inhibitory effect on migration of endothelial cells compared to zotarolimus.

Miura K, Nakaya H, Kobayashi Y. Experimental assessment of effects of

antiproliferative drugs of drug-eluting stents on endothelial cells. Cardiovascular

Revascularization Medicine. 2015;16:344-7