© 2014, Wiley Periodicals, Inc. DOI: 10.1111/joic.12092

CORONARY ARTERY DISEASE Dynamic Observation of Artery Endothelial Cell Layers in Response to Bare Metal Stent and Paclitaxel‐Eluting Stent In Vitro YUQING LIU, M.D., 1 XIAN WANG, M.D., 1 and JINSONG LU, M.D. 2 From the 1Department of Cardiology, Dongzhimen Hospital, First Affiliated Hospital of Beijing University of Chinese Medicine, Beijing, China; and 2Department of Pharmacology, Dongzhimen Hospital, First Affiliated Hospital of Beijing University of Chinese Medicine, Beijing, China

Objectives: We aimed to investigate the mechanism of paclitaxel‐eluting stent (PES) induced thrombosis by real‐time dynamic monitoring of live endothelial cells. Background: PES is widely used in clinics to inhibit restenosis effectively. However, late stent thrombosis is a major concern with the use of PES. Methods: We established an in vitro cell culture system to mimic the close contact of endothelial cells with PES struts. The dynamic response of porcine innominate artery endothelial cells (PIECs) to stents was observed using a bright field microscope and a high‐resolution charge‐coupled device Results: PES changed elongated PIECs to round PIECs within 24 hours. Paracellular gaps were readily observed in a PIEC monolayer exposed to PES. By contrast, paracellular gaps were almost undetectable in an endothelial monolayer incubated with bare metal stent (BMS). As incubation time was prolonged (days 5–9), round PIECs returned to their elongated shape, but paracellular gaps were retained at lower frequencies and smaller sizes than those on days 1 and 2. In addition, the PIEC monolayer in the PES group retained an uneven surface topology during incubation, whereas PIECs in the BMS group developed a smooth surface of epithelial cell sheets on days 5–7. Conclusion: Our findings showed that the shift of cell shape causes impaired integrity of the monolayer characteristic with enlarged paracellular gaps and uneven surface topology in exposure to PES. The results might serve as structural information for understanding the mechanism of PES‐induced early and late thrombosis. (J Interven Cardiol 2014;27:182–190)

Introduction Paclitaxel‐eluting stents (PESs) have been widely used in clinics to treat coronary artery diseases. PES effectively inhibits neointimal hyperplasia and exhibits acceptable safety when accompanied with antiplatelet therapy.1–3 However, stent thrombosis remains a severe concern after stent implantation owing to its high morbidity and mortality. Since PES was introduced, many interventional centers have reported late stent

Grant sponsor: Basic Scientific Research Foundation of Beijing University of Chinese Medicine. Address for reprints: Jinsong Lu, Dongzhimen Hospital, First Affiliated Hospital of Beijing University of Chinese Medicine, Beijing 100700, China. Fax: 86‐10‐82640224; e‐mail: jinsonglu_ [email protected]

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thrombosis up to 3 years after implantation, a phenomenon rarely observed in bare metal stents (BMS).4–6 The mechanism of PES‐induced thrombosis is not fully understood. Human autopsy studies have revealed that late stent thrombosis is associated with delayed arterial healing characterized by delayed reendothelialization and persistent fibrin deposition.6–8 Well‐established methods used to evaluate reendothelialization include histopathology, optical coherence tomography (OCT), and scanning electron microscopy (SEM), among others. OCT has become the standard method used to evaluate artery healing and analyze stent‐strut coverage in detail.9 SEM analyses provide detailed three‐dimensional information of an endothelial surface at a high resolution.10,11 However, such methods cannot allow a dynamic observation on the response of

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epithelial cells to stents in real time; thus, informative data about endothelial cells adaption to PES underlying a vulnerable position to thrombosis might be lost. Thus we developed a simplified and reliable method to mimic the close contact of endothelial cells with the struts of stents in vitro. We aimed to observe the immediate alteration of endothelial cells to exposure of PES and also the prolonged adaption of the same population of cells to PES. With the help of a bright‐ field microscope and high‐resolution charge‐coupled device (CCD), we observed the dynamic response of endothelial cells in a pre‐formed monolayer to PES and BMS, which were fixed into the tissue culture chamber.

Materials and Methods Cell Culture. Porcine innominate artery endothelial cells (PIECs) were purchased from the National Platform of Experimental Cell Resources for Sci‐Tech (Shanghai, China) and cultured according to the manufacturer’s instructions. In brief, cell suspensions (1–3  106 cells/ml) were seeded on 12‐well tissue culture plates and then cultured in complete DMEM medium supplemented with 7.5% FBS (Gibco, Carlsbad, CA, USA) and 1 antibiotics (Gibco) at 37 °C in a CO2 incubator. The medium was refreshed every 12 hours to elute the coating drug. The cells were used for less than 4 to 6 passages. Stent Placement. Commercially available 316 L steel stents (2 mm  12 mm) were used. Two kinds of drug‐eluting stent were purchased (Lepu, Changping, Beijing, China). For PES, paclitaxel concentration was 0.9 mg/mm2. A co‐coating stent contained 0.9 mg/mm2 of paclitaxel plus 0.5 mg/mm2 of Hirudin. The paclitaxel and Hirudin co‐eluting stents (PHESs) were included in this study to perform preliminary evaluation of the effect of co‐coating Hirudin on the effect of paclitaxel on epithelial cells. A permanent double‐sided tape (Scotch, St. Paul, MN, USA) was used to fix the stent in a cultural chamber. The tape was irradiated with UV for approximately 30 minutes on each side in a biosafety hood before use. The cells were then removed from the medium, and the middle part of the stent was gently adhered to the sterile tape. Afterward, the stent was placed firmly onto the cultured cells by attaching the ends of the tape on the inner lateral wall of the well. This method allowed the cell populations to reside at a constant distance from the struts of the stent during

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observation, which represents the in vivo relationship of endothelial cells with stents. This method is also a simplified approach of handling the culture conveniently to avoid stent movements. Imaging and Data Analysis. Imaging was performed by bright field microscopy under an inverted Olympus IX70 microscope equipped with a back‐ illuminated high‐resolution CCD camera (Andor Technology, Springvale, Belfast, UK). A CO2 incubation system (TOKAI HIT, Shizuoka‐ken, Japan) was placed on the stage of the microscope. The whole course of live cell imaging was performed at 37 °C in 5% CO2. These factors are important to maintain the physiological cellular environment. The cells were randomly selected from areas covered with stents and then examined. An objective (10 or 20) was used to acquire the cell images. The images were analyzed automatically using homemade software adapted with ImageJ and checked manually to confirm the results as described previously.12 The horizontal distance and the vertical height data from the images were extracted and plotted with Origin 8. Briefly, the images were independently taken by 2 observers. The pictures were then combined for each treatment group. An experienced investigator who was blinded to the treatment measured cell long axis, short axis, and height information using computer and in‐ house software. Eight images, including approximately 3,000 cells from 8 areas of interest from each group, were randomly chosen to be formatted and processed by the software. The boundaries of cells with clear body contours were highlighted. Measurement of cell body values was performed simultaneously by the software. Cell amount in the range of 200–500 was selected to measure morphological data, followed by analysis. When the sample number is less than 200 cells, the variance and error in the resulting data increase significantly. A sample number that is more than 500 is beyond the process ability of the software. Thus, a sample number between 200 and 500 was selected in each repeated experiment. All of the experiments were repeated 3 to 4 times independently.

Results Morphology of Subconfluent PIECs in Response to PES, PHES, and BMS. The sub‐confluent PIECs had an elongated shape with a narrow cell body and 2 long, thin ends that were several tens of micrometers

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long as measured on a tissue culture plate. After PIECs were exposed to BMS, the morphology remained unchanged and the cells continuously proliferated (Fig. 1A and B). Elongated PIECs changed to round PIECs and lost the long thin process in 1 hour after PES and PHES were added. Afterward, the cells

retained their abnormal shape until the end of the experiment (Fig. 1A). The confluent PIECs continued to amplify and subsequently developed multilayer sheets after 48 hours in the BMS group, as shown in the image analysis of the cell boundaries. These boundaries were blurred when 2 or more layers were captured

Figure 1. Morphological alteration of sub‐confluent endothelial cells to PES, PHES, and BMS. (A) Images of PIEC cells taken at different times after incubation with individual stents. The black region in the images is the struts of the stents. The cells in the BMS group are elongated and continued to proliferate, whereas the cells exposed to PES (Taxol) and PHES (Taxol þ Hirudin) are round and ceased to proliferate. (B) Proliferation curve of PIEC exposed to PES, PHES, and BMS during observation. (C) Amount of cells undergoing mitotic arrest state during observation. The mitotically arrested cells are characterized as round, bright cells detached from the bottom surface of the plate. Data are plotted as mean  standard deviation (SD). The amount of cells was plotted as the number of cells per 160 mm  160 mm area. Scale bar ¼ 100 mm.

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Figure 2. Continued.

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3 Figure 2. Morphological changes in PIEC monolayers in response to PES, PHES, and BMS. (A) Images of PIEC monolayers taken at different times after incubation with individual stents. No difference in cellular morphology was observed between the samples from days 5 to 9; thus, only the samples on day 5 are shown. On day 1, the cells were round instead of elongated in PES (Taxol) and PHES (Taxol þ Hirudin) groups. The PIEC cells partially returned to their elongated shape from days 3 to 5 in PES and PHES groups. The cells in the BMS group continued to proliferate from days 1 to 5. Multilayer sheets with a smooth surface were developed on day 4 of incubation. (B) Number of cells undergoing mitotic arrest at different times of incubation. The cells were round and detached from the monolayers. Data are plotted as mean  SD. The number of cells per 320 mm  320 mm area is plotted. (C) Cell layers were formed during observation. Monolayers were retained in PES and PHES groups from days 1 to 9. Multilayers were developed from days 3 to 9 in the BMS group. Scale bar ¼ 100 mm.

under the bright field microscope because their cell bodies overlapped. In PES and PHES groups, the cells stopped to proliferate (Fig. 1B), and some cells were morphologically circular. These cells were possibly in a mitotic arrest state until the end of the experiment (Fig. 1C). This result is consistent with that in previous studies, in which paclitaxel induces the cells to change in shape (from elongated to round) and undergo a cell mitotic arrest.13–15 Enlarged Paracellular Gaps Detected in Confluent Cell Monolayers During the Early Periods of PES and PHES Exposures But Not to BMS Exposure. The PIECs were allowed to amplify and develop a confluent monolayer to study the endothelial cell reactions in response to stent. The cells were seeded at a density of approximately 3  106 cells/ml and then cultured overnight. The stent with or without drug coatings was then fixed onto the monolayers. BMS did not alter the shape and the proliferation of the cells. PIECs exposed to BMS developed multilayer sheets with a smooth surface around the BMS struts (Fig. 2A). By contrast, PES and PHES stents caused PIECs to change morphologically from elongated to round within 24 hours. This change in shape produced gaps between endothelial cells with an average gap size of approximately 2.4 mm2 compared with the neglectable gaps (1 mm2 decreased from 35 to 8 per 0.16 mm2 area from days 1 to 7 in PES and PHES groups (Fig. 4A). The gaps were rarely observed in the BMS group at the time of observation (Fig. 4A). Figure 4C shows the detailed statistical data about the size and the frequency of the gaps in the sample obtained on day 1 from all of the

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groups. After exposure to PES and PHES, round PIECs returned partially to an elongated shape on days 5–7, indicating that the cells adapted to the drugs. The ratio of the long axis to the short axis (defined as cell shape index) of cells is plotted in Figure 4B to show the significant difference between BMS and PES (as well as PHES) on day 1 after the stents were added. Figure 4B also shows the tendency to regain the original cell morphology as incubation time was prolonged from days 3 to 7. The ratios of the cell shape index of 5.4 for PIECs in the BMS group and 3.9 for PIECs in PES and PHES groups were observed on days 5–7 (Fig. 4B). Uneven Epithelial Cell Surface Is Still Observed after Incubation with PES or PHES. Elongated PIECs with implanted PES or PHES changed to round PIECs. Aside from the horizontal measurements of the cell parameters, the height of PIECs in all of the groups was recorded. The highest point minus the lowest point along the Z‐axis from 1 cell body was defined as the range in height of the cell surface in our measurement. We noticed that the confluent PIEC monolayer in the BMS group continued to amplify and subsequently developed a smooth surface on days 3–7 (Fig. 5A). The height in the BMS group was 1 mm2 decreased from days 1 to 7. Data are presented as mean  SD per 0.4 mm  0.4 mm area. (B) PIEC cells changed in shape during observation. Elongated cells became round on day 1 in PES (Taxol) and PHES (Taxol þ Hirudin) groups. The round cells returned to their elongated shapes partially from days 3 to 7. The index of the cell shape is defined as the ratio of the long axis to the short axis of cells. Data are plotted as mean  SEM. (C) Frequency and size of the paracellular gaps in samples from BMS, PES, and PHES groups obtained on day 1. The average sizes of the gaps are evidently enlarged in PES and PHES groups compared with those in the BMS group.

were observed in samples obtained on day 90 comparing with day 30, and a few gaps were still detected in samples obtained on day 180, if these are not the coincidental artifacts introduced by sample preparation for SEM. Based on identical process procedure performed on all of the samples in their study, the accidental artifacts correlated with the date of incubation are unlikely to occur. These in vivo data are consistent with our observation in vitro. However, more detailed SEM evaluations with the aim to check paracellular gaps and topology of endothelium are needed to identify the abnormality of endothelium in vivo postimplantation of PES. The pharmacological effect of paclitaxel is extensively investigated.21,22 It shifts the dynamic state of microtubule to a stable state, which not only alters cells shape but also impairs cells movement.13,23 Considering the special case of endothelial cells, more requirements of cellular flexibility and dynamic fluctuation of the cell body are needed to maintain the integrity of the vessel walls during continuous dilation and contraction of arteries compared with other cells types in a still environment. Thus, paclitaxel‐stabilized cytoskeleton

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may also possibly impair the dynamic fluctuation of an endothelial cell body and may interfere with the integrity of the endothelium transiently. These factors may contribute to the formation of thrombosis. As such, new biophysical and cell biological investigations are required to determine the exact function of paclitaxel in stent thrombosis. The present study provided a new perspective to study the response of endothelial cells to PES with more detailed dynamic information. Other cell biological investigations in vitro may use this system to obtain direct dynamic information of molecular changes using genetically engineered fluorescent markers. Although in vivo and in vitro results as well as various species are different, our data clearly showed an abnormal morphology of endothelial cell monolayers in response to PES and PHES, which might serve as a hint to investigate alteration in endothelium in vivo. The present study is the first to address a structural issue involving paracellular gaps and topology of the endothelial surface to understand the mechanism of paclitaxel‐induced thrombosis, which is worth further cell biological and biomedical investigations.

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Figure 5. Surface topology and cellular height of the PIEC cell layers exposed to PES, PHES, and BMS. (A) Enlarged images of the surface of PIEC layers. The smooth surface was observed in BMS group (left) with all of the surface membranes almost found in the same focal plane. Elongated cells were found in the monolayers of PIEC in PES (middle) and PHES (right) groups. (B) Frequency and height of the cell in samples from BMS, PES, and PHES groups on day 7. The average heights of the cells increased in PES and PHES groups compared with those in the BMS group. Scale bar ¼ 30 mm.

Figure 6. Schematic diagram of the proposed mechanism of PES‐induced abnormality in endothelial cell monolayers. The healthy endothelial cells have a narrow and plane shape that develops a monolayer with an intact and smooth surface (orange cells). Paclitaxel‐poisoned endothelial cells are round, in which the assembly of these cells has paracellular gaps and an uneven monolayer surface (purple cells).

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Limitations The present study is an in vitro investigation that used cultured porcine artery endothelial cells. The results may not correlate with endothelium reaction in vivo. The species difference between human and porcine is another issue that may restrict the relevance of the data to clinical outcome. Moreover, the observation time was relatively short and long‐term studies should be conducted.

Conclusion This study demonstrated that paclitaxel induced morphology alteration of PIECs from elongated to round, which causes impaired integrity and altered smoothness of the PIEC monolayer characteristic with enlarged paracellular gaps and uneven monolayer surface topology in exposure to PES in vitro. The results indicate possible structural information for probing the mechanism of PES‐induced early and late thrombosis.

Acknowledgments: This study was supported by Basic Scientific Research Foundation of Beijing University of Chinese Medicine. We thank Dr. Hui Li of Chinese Academy of Science for data analysis and critical discussion of the manuscript.

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Journal of Interventional Cardiology

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Dynamic observation of artery endothelial cell layers in response to bare metal stent and paclitaxel-eluting stent in vitro.

We aimed to investigate the mechanism of paclitaxel-eluting stent (PES) induced thrombosis by real-time dynamic monitoring of live endothelial cells...
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